Microrna-198 as a tumor suppressor in pancreatic cancer

ABSTRACT

A novel network of tumorigenic prognostic factors is identified that plays a critical role in advanced pancreatic cancer (PC) pathogenesis. This interactome is interconnected through a central tumor suppressive microRNA, miR-198, which is able to both directly and indirectly modulate expression of the various members of this network to alter the molecular makeup of pancreatic tumors, with important clinical implications. When this tumor signature network is intact, miR-198 expression is reduced and patient survival is dismal; patients with higher miR-198 present an altered tumor signature network, better prognosis and increased survival. Further, according to the present disclosure, MiR-198 replacement reverses tumorigenicity in vitro and in vivo. As such, an embodiment of the disclosure is a method of treating cancer in an individual, comprising the step of increasing the level of active microRNA-198 molecules in the pancreatic cancer tumor cells of the individual by an amount sufficient to cause an improvement in the pancreatic cancer in the individual

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/567,852 filed Aug. 6, 2012, which claims the benefit of priority toU.S. Provisional Patent Application No. 61/515,416 filed on Aug. 5,2011, both of which are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. CA140828awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The field of the invention regards at least cell biology, molecularbiology, and medicine. In specific cases, the field of the inventionincludes pancreatic cancer.

BACKGROUND OF THE INVENTION

Pancreatic cancer (PC) is the fourth leading cause of cancer-relateddeaths in North America, with a <6% five-year survival prognosis (Jemalet al., 2010; Li et al., 2008; Zhang et al., 2010). Understanding themechanisms that give rise to PC pathogenesis and identifying prognosticmarker signatures are critical for the development of new diagnostic andtherapeutic strategies. The complex biological functions that give riseto cancer pathogenesis can rarely be attributed to individual moleculesbut rather arise from key interactions among various heterogeneouscomponents interacting in modular regulatory networks, which result in aspecific disease signature with far-reaching clinical effects (Bonnet etal., 2010; Hartwell et al., 1999). Described below are a multitude ofindividual molecules whose interactions were previously unknown, but areelucidate in the Detailed Summary.

Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in ˜90%of human pancreatic adenocarcinomas (Argani et al., 2001; Bharadwaj etal., 2008; Li et al., 2008; Muminova et al., 2004). It was previouslyreported that MSLN overexpression leads to increased PC cellproliferation, invasion, and migration in vitro and increased tumorgrowth in vivo (Li et al., 2008). Yet little is known about themechanisms through which MSLN overexpression gives rise to thisaggressive phenotype. Further, several studies have identified roles formicroRNAs (miRNAs) in PC pathogenesis. MiRNAs are a group of small,non-coding RNA molecules that act as posttranscriptional regulators ofmessenger RNA activity and are frequently dysregulated in cancers(Garofalo et al., 2009). It has been postulated that a single miRNA canregulate several links of an entire functional network, and itsdysregulation can therefore give rise to a complex disease phenotype(Wang et al., 2011). It was previously reported that MSLN constitutivelyactivates NF-κB and promotes cell survival (Bharadwaj et al., 2011a;Bharadwaj et al., 2011b). OCT-2 is a bi-functional TF that can exertboth activating and repressing functions in a context-dependent manner(Friedl and Matthias, 1995; Liu et al., 1996). Its expression waspreviously thought primarily restricted to B-cells. All tumor cell linesof the B-cell lineage express OCT-2, including Hodgkin's disease cells(Bargou et al., 1996) and non-Hodgkin's lymphoma (Pileri et al., 2003).ZEB1 is a crucial epithelial-to-mesenchymal transition (EMT) activatorin human colorectal and breast cancer (Burk et al), and has been linkedto increased EMT and chemoresistance in pancreatic cancers (Wang et al.,2009). ZEB1 also directly suppresses transcription of and is involved ina reciprocal regulatory loop with miRNAs in the miR-200 family (Burk etal, 2008). PBX-1 was initially identified as a participant in pre-B-cellacute lymphoblastic leukemia (Asahara et al., 1999; Dutta et al., 2001),has been associated with progression of melanoma, (Shiraishi et al.,2007) and is an inducer of the gene for VCP, a ubiquitously expressedprotein involved in cell survival (Wang et al., 2004). VCP is associatedwith cancer growth and is a prognostic marker for PC metastasis (Asai etal., 2002; Yamamoto et al., 2004c; Yamamoto et al., 2004d; Yamamoto etal., 2004e).

The approach of studying a single molecule in an effort to identifyeffective anti-tumor targets is quickly being replaced by a system-wideapproach to dissect the complex interactions between genes, RNA, andproteins in regulating tumor progression. Here, a unique perspective onthe interplay between several factors in a functional network ispresented, which approaches the study of the effects of a single,central microRNA from a network biology framework. This disclosureuncovers a novel functional interactome in PC, dissects the mechanismsthrough which a central miRNA can alter the molecular makeup ofpancreatic tumors, and identifies a pattern of expression thatcorrelates directly with patient prognosis and survival. Examined arethe expression, mechanisms of regulation, and resulting functions ofthis regulatory interactome, linking together the influence of variousseemingly heterogeneous tumorigenic factors as a modular unit in PCpathogenesis, with significant clinical implications. In additionclinically relevant treatments for PC are disclosed, as is a biomarkersignature which predicts treatment response and prognosis of patientswith PC.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method of treating cancer.Specifically, the method includes increasing the levels of miRNA-198 incancer cells in which high levels of MSLN, OCT-2, PBX-1, VCP, and/orZEB1 are expressed.

A general embodiment of the disclosure is a method of treating cancer inan individual, comprising the step of delivering to the individual aneffective amount of an agent that increases the level of microRNA-198molecules in cancer cells of the individual. To increase the levels, theagent may upregulate microRNA-198 and/or may directly deliver miRNA-198to the cancer cell. The agent may comprise microRNA-198, a microRNA-198mimic, and/or a modified microRNA-198. The cancer may be a cancer inwhich MSLN, OCT-2, PBX-1, VCP, and/or ZEB1 are upregulated and/orexpressed, such as pancreatic cancer, ovarian cancer, or liver cancer.

The composition may be administered through a variety of means. Forexample, the the composition is administered by a viral vector, anon-viral vector, a liposome, a microcarrier, nanocarrier, or acombination thereof. The composition may also be administered locally,systemically, or a combination thereof. In an embodiment of thedisclosure, the composition is delivered in a single or multiple cyclesof treatment.

In embodiments of the invention, delivering an effective amount of anagent that increases the level of microRNA-198 in cancer cells causes animprovement in the cancer by inhibiting migration, invasion,proliferation, tumor growth, metastatic potential, tumorigenesis or acombination thereof of the cancer and thereby increasing survival andprognosis of patients. The individual with cancer may be furtherprovided with one or more additional anti-cancer therapies, such aschemotherapy, radiotherapy, immunotherapy, gene therapy, surgery,non-microRNA-198 microRNA, siRNA or a combination thereof. Thechemotherapeutic agent effective against cancer may comprisegemcitabine, 5-fluorouracil, cisplatin, irinotecan, paclitaxel,capecitabine, oxaliplatin, streptozocin, or a combination thereof. ThemicroRNA-198 molecule level may be levels of modified microRNA-198,unmodified microRNA-198, microRNA-198 mimics, or a mixture thereof. Theagent may comprise an oligonucleotide that is 90%, 95% or 100% similarto SEQ ID NO: 1. Additionally, the method may further comprise the stepof determining the levels of MSLN, OCT-2, PBX-1, VCP, and/or ZEB1 in thecancer of the individual.

The method of claim 1, wherein the miRNA-198 comprises SEQ ID NO:1.

Another general embodiment of the disclosure is a method of inhibitingproliferation and metastatic potential of at least one cancer cell in anindividual, comprising delivering to the individual an effective amountof a composition which comprises an agent that increases the levels ofmicroRNA-198 molecules in a cancer cell. In one embodiment, the agentupregulates microRNA-198. The composition may be administered by a viralvector, a non-viral vector, a liposome, a viral vector, a microcarrieror a combination thereof. The composition may be administered locally,systemically, or a combination thereof. The agent may comprisemicroRNA-198, for example, SEQ ID NO:1, or the agent may comprise amodified microRNA-198 oligonucleotide or a microRNA-198 mimic. The agentmay comprise an oligonucleotide that is 90%, 95% or 100% similar to SEQID NO: 1. The individual may be further provided one or more additionalanti-cancer therapies, such as chemotherapy, radiotherapy,immunotherapy, gene therapy, surgery, non-microRNA-198 microRNA, siRNA,or a combination thereof. In an embodiment, the agent is delivered inmultiple cycles of treatment. The cancer may be a cancer in which MSLN,OCT-2, PBX-1, VCP, and/or ZEB1 are upregulated and/or expressed, such aspancreatic cancer, ovarian cancer, or liver cancer.

Another general embodiment of the invention is a kit for cancertreatment, said kit housed in a suitable container and comprising afirst anti-cancer agent that increases the levels of active microRNA-198molecules in a cell. In an embodiment of the disclosure, the firstanti-cancer agent upregulates microRNA-198 in a cell, such as a cancercell. The first agent may comprise microRNA-198, a microRNA-198 mimic, amodified microRNA-198, or a combination thereof. The agent may comprisemicroRNA-198, for example, SEQ ID NO:1. The agent may comprise anoligonucleotide that is 90%, 95% or 100% similar to SEQ ID NO: 1. Theagent may further comprise a vector, a microcarrier, or a liposome. Thekit may further comprise one or more additional anti-cancer agents, suchas a chemotherapeutic agent which may be effective against cancer. In anembodiment of the disclosure, the chemotherapeutic agent effectiveagainst cancer comprises gemcitabine, 5-fluorouracil, cisplatin,irinotecan, paclitaxel, capecitabine, oxaliplatin, streptozocin, or acombination thereof. The one or more additional anti-cancer agent maycomprises one or more radioisotopes. The cancer may be a cancer in whichMSLN, OCT-2, PBX-1, VCP, and/or ZEB1 are upregulated and/or expressed,such as pancreatic cancer, ovarian cancer, or liver cancer.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing.

FIG. 1 illustrates that the interactome of tumorigenic factorsinterconnected through miR-198 serves as a prognostic indicator of PC.FIG. 1A is the profiling of 95 cancer-associated miRNAs in MIA-V vsMIA-MSLN cells. FIG. 1B is a graph of log₁₀ miR-198 levels for 37patients. FIG. 1C is a graph of the percent patient survival after thetime of diagnosis for patients in the miR-198 low and miR-198 highgroups. FIG. 1D is a linear regression analysis correlating miR-198expression to mRNA levels of FSTL1, OCT-2, MSLN, PBX-1, and VCP inpatient tumor tissues. FIGS. 1E-1F are five-order Venn diagramrepresenting the complex interactome between the factors in the network.The percent of patients in which the specific factors are upregulated(or downregulated in the case of miR-198) either individually or for allpossible combinations is shown for all patients in FIG. 1E, for patientsin the miR-198-Low group alone in FIG. 1F, or for patients in themiR-198-High group alone in FIG. 1G.

FIG. 2 illustrates MSLN regulates miR-198 expression throughNF-κB-mediated induction of OCT-2. FIG. 2A is a graph of miR-198 levelsfold change over a control in HPDE-V and HPDE-MSLN cells. FIG. 2B is themiR-198 expression fold change over and demonstrates that silencing MSLNrestores miR-198 expression. FIG. 2C is a graph illustrating miR-198expression and MSLN mRNA levels in a variety of different types ofcells. Fold change was calculated relative to PC cell with lowestexpression of each factor. FIG. 2D shows a linear regression analysis ofFSTL1 expression versus miR-198 levels in a panel of PC cells showing asignificant positive correlation (p<0.001, R²=0.87). FIG. 2E is a graphwhich shows FSTL1 mRNA levels and demonstrates FSTL1 expression isdecreased in MIA-PaCa2 cells in accordance with miR-198 expressionfollowing forced MSLN expression. FIG. 2F illustrates that wedelolactonetreatment restores miR-198 expression in MIA-MSLN cells to pre-MSLNlevels and FIG. 2G illustrates wedelolactone treatment blocks OCT-2induction in MIA-MSLN cells. FIG. 2H illustrates ShRNA-mediatedsilencing of OCT-2 rescues miR-198 expression in MIA-MSLN cells.

FIG. 3 illustrates that MiR-198 is the central link between upstreamregulatory factors MSLN and OCT-2 and the closely correlated downstreamPBX-1/VCP tumorigenic axis. FIG. 3A demonstrates MSLN protein expressioncorrelates positively with PBX-1 expression, and both correlatenegatively with miR-198 (p<0.005), in a PC cell line panel. FIG. 3Bdemonstrates increased OCT-2 protein expression is accompanied by astrong induction in PBX-1. FIG. 3C demonstrates VCP expression, alongwith downregulation in miR-198 expression. FIG. 3D demonstrates PBX-1expression increases following MSLN overexpression in MIA-PaCa2 cells,and is restored following miR-198 overexpression. FIG. 3E demonstratesVCP expression in AsPC1 cells is downregulated ˜50 fold followingmiR-198 overexpression. FIG. 3F demonstrates Wedelolactone results in ablock in PBX-1 expression. FIG. 3G is a dual-luciferase reporter assaywhich shows a ˜65% reduction in luciferase expression of a PBX-1 3′UTRor in FIG. 3H a ˜70% reduction in luciferase expression of a VCP 3′UTRluciferase reporter following miR-198 overexpression, which is abolishedwhen the miR-198 target site is mutated and/or deleted. Expressed asfirefly/Renilla ratio.

FIG. 4 illustrates miR-198 reciprocally regulates MSLN expression bybinding to target sites within the MSLN CDS. FIG. 4A demonstratesMiR-198 overexpression blocks MSLN at the protein level. FIG. 4Bdemonstrates MSLN mRNA levels are partially downregulated followingmiR-198 overexpression. FIG. 4C demonstrates MiR-198 reduces expressionof a co-transfected MSLN expression plasmid at the protein level, with apartial decrease in mRNA expression. FIG. 4D illustrates site-directedmutagenesis of each of the three miR-198 binding sites within the MSLNcoding region separately or in combination leads to differentialrestoration MSLN protein expression in the presence of miR-198. FIG. 4Edemonstrates MiR-198 decreases luciferase expression in WT MSLN CDSconstructs for sites 2 and 3 (p<0.05) but not significantly for site 1.Mutating the miR-198 seed region for sites 2 or 3 restores luciferaseexpression. Expressed as firefly/Renilla ratio. Mean±SD.

FIG. 5 illustrates stable miR-198 reconstitution reduces the tumorigenicfunctions of mesothelin-overexpressing pancreatic cancer cells in vitro:FIG. 5A demonstrates MSLN overexpression increases MIA-PaCa2 cellmigration, and this effect is reversed following miR-198 overexpression(p<0.05). FIG. 5B demonstrates average proliferation (p<0.05) and FIG.5C demonstrates invasion of AsPC1 cells is decreased ˜50% followingmiR-198 overexpression (p<0.05). FIG. 5D illustrates wound healing assayshows that miR-198 decreases the migratory potential of MIA-MSLN cells.FIG. 5E demonstrates MiR-198 overexpression reduces the ability ofMIA-MSLN cells for anchorage independent growth in soft agar. FIG. 5Fillustrates the use of an antisense inhibitor of miR-198 leads tosignificantly increased proliferative (p<0.05) and FIG. 5G illustratesmigratory potential of MIA-V cells (p<0.05). FIG. 5H demonstrates PBX-1silencing has a significant (p<0.05) but modest effect on proliferation.FIG. 5I demonstrates PBX-1 overespression in MIA-V or MIA-MSLN-miR-198cells results in an increase in migration resembling that observed inMIA-MSLN cells (p<0.05). PBX-1 silencing reduces the MSLN-mediatedincrease in migration (p<0.05). Mean±SD.

FIG. 6 illustrates miR-198 is an antagonist of MSLN-mediated autocrinePC cell survival and resistance to TNF-α-induced apoptosis. FIG. 6Aillustrates a TUNEL assay which shows a significant increase inapoptosis after TNF-α, treatment in two high MSLN cells followingoverexpression of miR-198. FIG. 6B demonstrates a TUNEL assay whichshows a significant decrease in apoptosis in MIA-V cells (high miR-198cells) down to MIA-MSLN cell levels following blocking of miR-198(MIA-V-Zip-198). FIG. 6C demonstrates overexpression of miR-198 inMIA-MSLN cells results in caspase 3 cleavage.

FIG. 7 illustrates miR-198 overexpression modulates expression ofregulatory network and reduces tumor growth and metastatic spread invivo. FIG. 7A demonstrates nude mice injected s.c. withMIA-MSLN-miR-Ctrl cells started to develop tumors by 7 days postinjection. The mice were sacrificed at 25 days post injection when theirtumors reached an average volume of 2000 mm³. Only 3 of 9 mice injectedwith MIA-MSLN-miR-198 cells developed tumors, with an average volume ofonly 36 mm³ by day 45 post injection. FIG. 7B are representative imagesfor each s.c. mouse group, 9 mice per group. FIG. 7C shows nude miceinjected orthotopically with MIA-MSLN-miR-Ctrl cells developed primarytumors after 4 weeks that were approximately 10-fold larger (by weight)than tumors primary tumors developed by mice injected withMIA-MSLN-miR-198 cells, and included 8 mice per group. FIG. 7D showsprimary tumors resected from each mouse in both groups. FIG. 7E showsGFP expression in the tumor cells allows for visualization of tumorspread. FIG. 7F shows real-time RT-PCR was used to confirm RNA levels ofall the factors in the regulatory network. Mean±SD, n=4, *p<0.05.

FIG. 8 is a diagram of the network of heterogeneous prognostic factorsfor pancreatic cancer interconnected through modulation of centraltumor-suppressive miR-198.

FIG. 9 illustrates miR-198 modulation in stable cell lines. FIG. 9Arepresents real-time RT-PCR, which shows the mRNA levels of all thefactors in the proposed network (MSLN, OCT-2, PBX-1, VCP, and FSTL1,respectively), segregated into two groups based on miR-198 levels asdescribed in FIG. 1. FIG. 9B is a linear regression analyses whichdepicts the correlations between each factor and its counterparts.Separate regression analyses were performed for MSLN, OCT-2, PBX-1, VCP,and FSTL1, respectively.

FIG. 10 illustrates PBX-1 and VCP are predicted targets for miR-198.FIG. 10A demonstrates PBX-1 has a predicted 8mer binding site formiR-198 in its 3′UTR. FIG. 10B demonstrates VCP is also a predictedtarget for miR-198, with a predicted 8mer binding site for miR-198 inits 3′UTR. FIG. 10C demonstrates the predicted binding site for miR-198in the 3′UTR of PBX-1 and FIG. 10D demonstrates VCP is evolutionarilyconserved in a majority of species examined using TargetScan software.FIG. 10E is a graph of real-time RT-PCR for MSLN and PBX-1 in a panel ofPC cell lines, which shows that PBX-1 increases as MSLN expressionincreases. Fold change was calculated relative to PC cell with lowestexpression. Mean shown. FIG. 10F demonstrates PBX-1 levels increase asmiR-198 levels decrease in a panel of PC cell lines. Fold change wascalculated relative to PC cell with lowest expression. Mean shown. FIG.10G demonstrates Mir-198 downregulates PBX-1 mRNA in MIA-MSLN cells andFIG. 10H in ASPC1 cells. FIG. 10I demonstrates MIA-MSLN cells show a˜2-fold increase in VCP mRNA levels over MIA-V cells. MiR-198overexpression reduces VCP down to MIA-V cell levels. FIG. 10Jdemonstrates VCP mRNA levels are downregulated ˜5 fold in ASPC1 cellsfollowing miR-198 overexpression. FIG. 10K is a schematic representationof the construction scheme for the miR-198 binding site within the VCP3′UTR. The restriction sites including XbaI and BamH1 and additionalsites used for insert confirmation are shown, along with the3-nucleotide mutation incorporated into the miR-198 seed region. FIG.10L are nucleotide sequences for the first ˜500 bases of the PBX-1 3′UTRthat were cloned into the PSGG-3′UTR vector for luciferase assays,including the WT, point mutation, and deletion mutations in the miR-198binding site seed regions (bold and highlighted).

FIG. 11 illustrates additional evidence that MSLN-mediated NF-κBactivation induces OCT-2 expression. FIG. 11A illustrates OCT-2expression shown in MIA-V and MIA-MSLN cells. FIG. 11B shows OCT-2expression is blocked following Wedelolactone treatment of AsPC1 cells.

FIG. 12 illustrates miR-198 modulation in stable cell lines. Real-timeRT-PCR was used to confirm miR-198 levels—FIG. 12A: miR-198overexpressing MIA-MSLN cells, FIG. 12B: miR-198 overexpressing ASPC1cells, FIG. 12C: miR-198 overexpressing and/or Zip-198 blocked MIA-Vcells, and FIG. 12D: miR-198 overexpressing or FIG. 12E: Zip-198 blockedHPDE stable cell lines.

FIG. 13 are the construct details for miR-198 target analysis of MSLNCDS. FIG. 13A illustrates miR-198 has three target sites in the MSLNgene, as predicted by RNA22 software. FIG. 13B shows three nucleotidesubstitutions were introduced via site-directed mutagenesis into each ofthe 3 predicted binding sites for miR-198 in the MSLN coding region. Themutations were selected so as to not alter the amino acid sequence ofthe MSLN protein to allow for proper expression and detection whilestill preventing miR-198 directed targeting. Seed region are shown inbold. FIG. 13C shows miR-198 levels shown in COS-7 cells followingtransfection of miR-198 expression plasmid. FIG. 13D is a schematicrepresentation of the construction scheme for miR-198 binding site 3within the MSLN coding region. The restriction sites including XbaI andBamH1 and additional sites used for insert confirmation are shown, alongwith the 3-nucleotide mutations incorporated into the miR-198 seedregion. The same scheme was used in all six constructs.

FIG. 14 illustrates additional evidence of miR-198 tumorigenic functionsin PC cells in vitro. FIG. 14A is a monolayer wound-healing assay whichshows a reduction in migration and proliferation back to MIA-V controllevels following miR-198 precursor transfection compared to a scrambledmiRNA control. Mean±SD. FIG. 14B demonstrates invasion is reduced by 24%36 h after miR-198 precursor transfection (p<0.05). Mean±SD. FIG. 14Cdemonstrates migration is reduced by 44% 36 h after transient miR-198transfection (p<0.05). Mean±SD. FIG. 14D shows MiR-198 overexpressiondecreases migration of AsPC1 (p<0.05) cells by >50%. Mean±SD. FIG. 14Edemonstrates MiR-198 overexpression decreases proliferation (resultsshown as mean of five wells. Error bars omitted for clarity) and FIG.14F demonstrates invasion (across a matrigel matrix) of MIA-MSLN cells(p<0.05) FIG. 14G are monolayer wound healing assays which wereperformed in different serum conditions to demonstrate reduced migrationand proliferation of both MIA-MSLN and AsPC1 cells following miR-198overexpression. FIG. 14H demonstrates blocking miR-198 in HPDE cellsresults in increased migration after 72 h (p<0.05). Furtheroverexpression of miR-198 in these cells did not seem to have an effect.Mean±SD. FIG. 14I shows that blocking of miR-198 in HPDE cells led to anincrease in migration. FIG. 14J demonstrates modulation of PBX-1 inMIA-PaCa2 cells increases proliferation.

FIG. 15 illustrates evidence that ZEB1 induction represses miR-198following MSLN-mediated NF-κB activation. FIG. 15A shows ZEB1 expressionincreases following MSLN overexpression. FIG. 15B shows ShRNAs againstMSLN reduce ZEB1 expression at the mRNA and FIG. 15C. are protein levelsin MIA-MSLN cells. FIG. 15D illustrates Wedelolactone treatment blocksZEB1 expression in MIA-MSLN cells. FIG. 15E shows ZEB1 is expressed in amajority of cell lines with high MSLN/low miR-198 expression, while itis not expressed in miR-198 high Capan-1 or HPDE cells. Line depictsseparation between non-contiguous lanes. FIG. 15F. shows transienttransfection of shRNAs against ZEB1 reduces ZEB1 protein expression andrestores miR-198 expression in MIA-MSLN cells. Figure was modified toshow samples run on the same gel but in non-contiguous lanes, asdepicted by separation. FIG. 15G demonstrates ZEB1 transfection inMIA-PaCa2 cells reduces miR-198 expression after 48 h.

DETAILED DESCRIPTION OF THE INVENTION

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “synergistic” or “synergistically” as used herein refers to theaddition of two reactants that may or may not react in the same pathwaywith each other, from which the resulting product of the reactionproceeds to a further extent than one of skill in the art would predict.In a specific embodiment, two compounds act synergistically when theresult achieved upon using them in combination is greater than the sumof the results of the compounds when used separately.

The phrase “effective amount” as used herein means that amount of acompound, material, or composition comprising a compound of the presentinvention that is effective for producing some desired effect, e.g.,halting the growth of, reducing the size of, and/or causing apoptosis ina cancer cell. In one embodiment, the effective amount is enough toreduce or eliminate at least one cancer cell. One of skill in the artrecognizes that an amount may be considered effective even if the cancercell is not totally eradicated but decreased partially. For example, thespread of the cancer may be halted or reduced, a side effect from thecancer may be partially reduced or completed eliminated, and so forth.The effective amount may also be a therapeutically effective amount.

The terms “inhibit,” “inhibitory,” or “inhibitor” as used herein refersto one or more molecules that interfere at least in part with the growthor activity of the molecule or cell it inhibits. The inhibition of acancer cell may be the inhibition of growth of at least one cancer cell.

As used herein, “treat” and all its forms and tenses (including, forexample, treat, treating, treated, and treatment) refer to boththerapeutic treatment and prophylactic or preventative treatment. Thosein need thereof of treatment include those already with a pathologicalcondition of the invention (including, for example, a cancer) as well asthose in which a pathological condition of the invention is to beprevented. In certain embodiments, the terms “treating” and “treatment”as used herein refer to administering to a subject a therapeuticallyeffective amount of a composition so that the subject has an improvementin the disease or condition. The improvement is any observable ormeasurable improvement. Thus, one of skill in the art realizes that atreatment may improve the individual's condition, but may not be acomplete cure of the disease. Treating may also comprise treatingsubjects at risk of developing a disease and/or condition of theinvention.

As used herein the term “metastatic” (and all other forms and tenses,including, for example, metastasis, metastasize, etc.) when used aloneor in conjunction with cancer refers to the spread of a cancer from onepart of the body to another, unless otherwise indicated by the use orcontext. Typically, a tumor formed by cells that have spread is called a“metastatic tumor” or a “metastasis.” The metastatic tumor containscells that are like those in the original (primary) tumor.

As used herein, an “individual” is an appropriate individual for themethod of the present invention. A subject may be a mammal and inspecific embodiments is any member of the higher vertebrate classMammalia, including humans; characterized by live birth, body hair, andmammary glands in the female that secrete milk for feeding the young.Additionally, mammals are characterized by their ability to maintain aconstant body temperature despite changing climatic conditions. Examplesof mammals are humans, cats, dogs, cows, mice, rats, horses, sheep, pigsand chimpanzees. Subjects may also be referred to as “patients” or“subjects”.

An embodiment of the disclosure is the identification of a novel networkof tumorigenic prognostic factors that plays a critical role in advancedpancreatic cancer (PC) pathogenesis. This interactome is interconnectedthrough a central tumor suppressive microRNA, miR-198, which is able toboth directly and indirectly modulate expression of the various membersof this network to alter the molecular makeup of pancreatic tumors, withimportant clinical implications. When this tumor signature network isintact, miR-198 expression is reduced and patient survival is dismal;patients with higher miR-198 present an altered tumor signature network,better prognosis and increased survival. Further, according to thepresent disclosure, MiR-198 replacement reverses tumorigenicity in vitroand in vivo. This illustrates the therapeutics of attacking a complexheterogeneous network of factors through a central vantage point.

Another embodiment of the disclosure is miR-198 as a critical prognosticfactor for PC patient survival based on its expression in patienttumors. With miR-198 as a central vantage point, a systemic approach wasused to identify both novel and well-established PC prognostic factorsthat could either modulate or be modulated by miR-198. These studies aresummarized below, with a in-depth review in the example section. In aneffort to study the molecular interactions that lead to MSLN-mediatedpathogenesis in PC, the miRNA signature of MSLN-overexpressing vs. MSLNlow PC cells was examined. It was found that miR-198 was the mostsignificantly downregulated miRNA among a global dysregulation of miRNAsas a result of MSLN overexpression. Additionally, the MSLN-NF-κBinteraction was identified as a key component of the in the regulationof miR-198. Thus, an embodiment of the disclosure is a novel role forMSLN-mediated NF-κB activation as an indirect regulator of miR-198expression, through the induction of the repressive transcription factorOCT-2 (POU2F2) which was selected based on microarray analysis ofMSLN-overexpressing vs. MSLN low PC cells. It is also seen for the firsttime that OCT-2 is not only expressed in PC cells, but that it plays arole in regulating miR-198, and through this interaction also modulatesexpression of miR-198 downstream targets. Through indications found inthe microarray data, two downstream effectors of miR-198 wereidentified, Pre-B-cell leukemia homeobox factor 1 (PBX-1), andValosin-containing protein (VCP). Additionally, the PBX-1/VCP axis playsa key role in PC pathogenesis. Thus, this disclosure includes a uniqueperspective on the interplay between several factors in a functionalnetwork, approaching the study of the effects of a single, centralmicroRNA from a network biology framework. In doing so, a novelfunctional network was discovered in PC, as well as the mechanisms bywhich key interactions among tumor suppressive and tumor promotingmolecules can alter the molecular makeup of pancreatic tumors, withsignificant clinical implications. The functional network of prognosticfactors in pancreatic cancer by miR-198, illustrated in FIG. 8,contributes to increased tumor cell aggressiveness and decreased patientsurvival. MiR-198 acts as a tumor suppressor by interfering with thisfunctional network. In an embodiment of the invention, miR-198replacement therapy extends patient survival by leading to a change inthe molecular makeup of aggressive pancreatic tumors.

An embodiment of the invention is the therapeutic value of miR-198 andits influence on the interactions of an array of tumorigenic factors.There was a very clear distinction in the prognostic outcome of patientsfollowing resectable surgery. Patients with higher miR-198 levels had abetter prognosis, with 80% of them still alive at the 40 month mark. Instark contrast, only 11% of patients with low miR-198 levels survived tothis point. The causative role of miR-198 is demonstrated in thisincreased survival rate by showing that reconstitution of miR-198significantly reduces tumor growth and metastasis of PC cells in mousexenografts. In an embodiment of the disclosure, effects of miR-198 aredue not to the regulation of a single factor, but rather themodification of the molecular makeup of tumors through a concertedmodulation of a novel functional network of prognostic factors, whichincludes established PC biomarkers and effectors MSLN and NF-κB, as wellas factors previously uncharacterized in PC pathogenesis, OCT-2 and thePBX-1/VCP axis.

Within this concerted network, a complex reciprocal regulatory loop wasuncovered between MSLN and miR-198 that gives novel insight into themechanisms of MSLN-mediated PC pathogenesis. It was previously reportedthat MSLN overexpression leads to constitutive activation of NF-κB,resulting in a positive regulation of cell survival and proliferation ofPC cells under serum-reduced and anchorage-independent conditionsthrough NF-κB-mediated IL-6 induction (Bharadwaj et al., 2011a;Bharadwaj et al., 2011b). Here, these findings are extended to show thatMSLN-mediated NF-κB activation can result in modulation of miR-198expression through the induction of the POU domain TF OCT-2.

OCT-2 overexpression has been found in all types of tumors of the B-celllineage and OCT-2 has been associated with various hematologicmalignancies (Bargou et al., 1996; Heckman et al., 2006). Here, OCT-2was identified as an important factor in PC for the first time. OCT-2 isoverexpressed in a majority of PC cell lines and is upregulated in over80% of patient tumor tissues, and acts as a repressor of theFSTL1/miR-198 promoter which presents a novel role for this protein as aPC prognostic factor and functional target.

Another novel finding is that miR-198 can reciprocally target not justone, but multiple sites in the MSLN coding region, rather than its3′UTR, with an additive effect that leads to an almost complete block ofMSLN protein expression. While not bound by the hypothesis, this mayimply that dysregulation of MSLN or another component of theMSLN→NF-κB→OCT-2 pathway or other genomic insults affecting miR-198expression can result in downregulation of miR-198 and activation of afeed-forward mechanism resulting in MSLN overexpression. MiR-198effectively reverses the effects of MSLN in PC cells and tumors throughdirect modulation, and thus indirectly regulates NF-κB activation andOCT-2 induction, potentially altering a very vast array of tumorigenicproperties that define aggressive tumors.

In addition, miR-198 also targets the tumorigenic factors PBX-1 and VCP.PBX-1 dysregulation has been implicated in increased proliferation ofcancer cells (Park et al., 2008; Shiraishi et al., 2007); VCPoverexpression correlates with increased progression and metastaticpotential of a variety of cancers (Tsujimoto et al., 2004; Yamamoto etal., 2004a; Yamamoto et al., 2004b; Yamamoto et al., 2004c; Yamamoto etal., 2004d; Yamamoto et al., 2004e). However, this disclosuredemonstrates for the first time that modulation of PBX-1 in PC cellscontributes to MSLN-mediated proliferation. In addition, the resultsindicate a novel role for the PBX-1/VCP axis in PC cell migration, andimplicate PBX-1 as a factor responsible for the metastatic potential ofMSLN-overexpressing cells. Previous studies have linked the PBX-1/VCPaxis to metastatic potential and survival through the VCP-mediatedactivation of NF-κB (Asai et al., 2002). NF-κB also reciprocallymodulates PBX-1 and VCP expression through the regulation of miR-198 andthe interconnectivity of the interactome. VCP can thereby feed back intothe pathway, promoting maintenance of NF-κB activation, OCT-2 induction,and subsequent miR-198 repression.

The PBX-1/VCP axis is important in cell survival under cytokine stress,as demonstrated by the resistance of PBX-1/VCP overexpressing cells inTNF-α, mediated induction of apoptosis (Qiu et al., 2007). It wasreported that MSLN overexpressing cells are resistant to the apoptoticeffects of TNF-α, (Bharadwaj et al., 2011a), and here it is shown thatmiR-198 reconstitution can act as an antagonist of these increased cellsurvival effects. Taken together, the results show that miR-198 mediatedmodulation of the interactome has implications in preventingchemotherapeutic resistance of PC cells.

The examples identify an interactome of molecular entities that can beof prognostic value and provide insight into the diseased state of PCpatients. The dynamic behavior and function of a previouslyuncharacterized signaling network was elucidated which gives rise to anaggressive phenotype with clinical implications. The importance ofmiR-198 as a central component of this network is underscored by thesignificant reversal in tumorigenic aggressiveness that accompaniesmiR-198 reconstitution and the subsequent alteration in the molecularmakeup of tumors. In addition to elucidating the molecular mechanismsthrough which MSLN promotes pathogenesis, the examples below identifyOCT-2 and the PBX-1/VCP axis as critical biomarkers and targets for PCtreatment. By acting as the key regulator of this network, miR-198replacement therapy has the potential to influence the interactionsbetween these molecules and revert the most aggressive pancreatic tumorsto a more manageable, less invasive phenotype, and has wide reachingtherapeutic potential for other cancers where MSLN, OCT-2, PBX-1 or VCPplay important roles either individually or as part of this functionalnetwork.

Pancreatic Cancer

As the fourth leading cause of cancer-related deaths in North America,pancreatic cancer has the highest fatality rate of all cancers. Survivalstatistics are poor, because there are no reliable tests for earlydiagnosis and no effective therapies for the metastatic form ofpancreatic cancer (Landis et al., 1998; Torrisani and Buscail, 2002;Warshaw and Fernandez-del Castillo, 1992). By the time diagnosis ismade, the disease has usually spread to distant sites of the body.

Representative symptoms of pancreatic cancer include pain in the abdomenand back, loss of appetite, bloating, diarrhea or fatty bowel movements,and jaundice, for example. Diagnosis may be made on physical exam,abdominal ultrasound, and/or abdominal computed tomography, for example.A biopsy may be performed either percutaneously or endoscopically.Treatment is usually through chemotherapy, radiation therapy, andsurgery. The most commonly used chemotherapies are gemcitabine,fluorouracil, and capecitabine. The present invention may be employedwith any comventional treatment of PC, for example.

In an embodiment of the disclosure, levels of MSLN, OCT-2, PBX-1, VCP,ZEB1 or a combination thereof are measured in the cancer of theindividual. In an embodiment of the disclosure, either the levels of RNAor protein may be measured. In one example, if the levels of MSLN,OCT-2, PBX-1, VCP, and/or ZEB1 are high, then the individual is given ahigher dose of miRNA-198 therapy. If the levels of MSLN, OCT-2, PBX-1,VCP, and/or ZEB1 are low, then the patient prognosis and survival ispredicted to be high. While there are many variants of these proteinsand genes, as examples, the following accession numbers may be used:MSLN: NM_(—)005823; OCT2: NM_(—)002698; PBX-1: NM_(—)002585; VCP:NM_(—)025054; and ZEB1: NM_(—)001174094. An embodiment of the disclosureis a method of predicting patient outcome comprising the step ofmeasuring the protein or RNA levels of MSLN, OCT-2, PBX-1, VCP, and/orZEB1 in an individual with cancer.

miRNA-198 Therapy

MiRNA-198 therapy refers to a therapy that increases the level of activemicroRNA-198 molecules in a cell. The increase can come about bydirectly providing the microRNA-198 to a cell, or may come about byindirectly providing miRNA-198 to cell, such as through a vector. ThemiRNA-198 may be comprised on a RNA or DNA molecule that also comprisesadditional sequences. In some instances of the disclosure, the miRNA-198therapy is SEQ ID NO: 1. In another embodiment of the invention, themiRNA-198 is the seed region of miRNA-198 which is the first 12nucleotides of SEQ ID NO: 1. In other embodiments, the miRNA-198sequence differs from SEQ ID NO: 1 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or more nucleotides. In other embodiments, the miRNA-198 sequenceincludes an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or morenucleotides. In other embodiments, the miRNA-198 sequence includes thedeletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides.In some embodiments, the miRNA-198 will include one or more differentnucleotides, one or more deleted nucleotides, and/or one or moreinserted nucleotides.

The miRNA-198 therapy may also comprise a nucleic acid strand that iscomplimentary to miRNA-198. For example, the miRNA-198 complimentarystrand comprises 22 nucleotides that are complimentary to SEQ ID NO. 1.The complimentary strand may also only comprise 12 nucleotides that arecomplimentary to the seed sequence of miRNA-198. The complimentarystrand may also comprise at least one nucleotide sequence differencewhen compared with the true reverse complement sequence of the seedregion of the guide strand, wherein the at least one nucleotidedifference is located within nucleotide position 13 to the 3′ end ofsaid complimentary strand.

Strands or regions that are complementary may or may not be 100%complementary (“completely or fully complementary”), including to SEQ IDNO: 1. It is contemplated that sequences that are “complementary”include sequences that are at least about 50% complementary, and may beat least about 50%, 60%, 70%, 80%, or 90% complementary. In the range ofabout 50% to 70% complementarity, such sequences may be referred to as“very complementary,” while the range of greater than about 70% to lessthan complete complementarity can be referred to as “highlycomplementary.” Unless otherwise specified, sequences that are“complementary” include sequences that are “very complementary,” “highlycomplementary,” and “fully complementary.” It is also contemplated thatany embodiment discussed herein with respect to “complementary” strandsor region can be employed with specifically “fully complementary,”“highly complementary,” and/or “very complementary” strands or regions,and vice versa. Thus, it is contemplated that in some instances, asdemonstrated in the Examples, that siRNA generated from sequence basedon one organism may be used in a different organism to achieve RNAi ofthe cognate target gene. In other words, siRNA generated from a dsRNAthat corresponds to a human gene may be used in a mouse cell if there isthe requisite complementarity, as described above. Ultimately, therequisite threshold level of complementarity to achieve RNAi is dictatedby functional capability.

It is specifically contemplated that there may be mismatches in thecomplementary strands or regions. Mismatches may number at most or atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,residues or more, depending on the length of the complementarity region.

In some methods of the invention, miRNA and/or candidate miRNA moleculesor template nucleic acids may be isolated or purified prior to theirbeing used in a subsequent step. miRNA and/or candidate miRNA moleculesmay be isolated or purified prior to introduction into a cell.“Introduction” into a cell includes known methods of transfection,transduction, infection and other methods for introducing an expressionvector or a heterologous nucleic acid into a cell. A template nucleicacid or amplification primer may be isolated or purified prior to itbeing transcribed or amplified. Isolation or purification can beperformed by a number of methods known to those of skill in the art withrespect to nucleic acids. In some embodiments, a gel, such as an agaroseor acrylamide gel, is employed to isolate the miRNA and/or candidatemiRNA.

In various embodiments, miRNAs are encoded by expression constructs. Theexpression constructs may be obtained and introduced into a cell. Onceintroduced into the cell the expression construct is transcribed toproduce various miRNAs, such as miRNA-198. Expression constructs includenucleic acids that provide for the transcription of a particular nucleicacid. Expression constructs include plasmid DNA, linear expressionelements, circular expression elements, viral expression constructs, andthe like, all of which are contemplated as being used in thecompositions and methods of the present invention. In certainembodiments at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNAmolecules are encoded by a single expression construct. Expression ofthe miRNA molecules may be independently controlled by at least about 2,3, 4, 5, 6, 7, 8, 9, 10 or more promoter elements. In certainembodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreexpression constructs may introduced into the cell. Each expressionconstruct may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNAmolecules. In certain embodiments miRNA molecules may be encoded asexpression domains. Expression domains include a transcription controlelement, which may or may not be independent of other control orpromoter elements; a nucleic acid encoding an miRNA; and optionally atranscriptional termination element. In other words, an miRNA cocktailor pool may be encoded by a single or multiple expression constructs. Inparticular embodiments the expression construct is a plasmid expressionconstruct.

The delivery of the miRNA therapy may occur through several forms, suchas through encapsulation of a chemically modified or through anunmodified RNA moiety within a viral or non-viral delivery vessel.Non-viral deliver vessels include nanoparticles, microparticles,liposomes, etc., which may be targeted to a cancer site or systemic. ThemiRNA-198 therapy can also be delivered as a plasmid or minivector basedexpression system where it can then be expressed and processed by theRNAi machinery in cells to form a mature miR-198 form or a derivativethereof. In an embodiment of the invention, a nanoparticle based,targeted delivery system of encapsulated miR-198 oligonucleotide and/ora plasmid expressing miR-198 is utilized.

Nucleic Acid-Based Expression Systems

In certain aspects of the invention, an agent comprising a nucleic acid,such as miRNA-198 is employed. Such an agent may be comprised within anexpression system, such as on a vector, although alternatively the agentis not comprised within an expression system.

Vectors

Nucleic acids of the invention, particularly DNA templates or DNAconstructs for miRNA expression, may be produced recombinantly. Proteinand polypeptides may be encoded by a nucleic acid molecule comprised ina vector. The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques (see, for example, Maniatis et al., 1988 andAusubel et al., 1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with 13galactosidase, ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Non-limiting examples of virus vectors that may beused to deliver a nucleic acid of the present invention are describedbelow.

Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the present invention asit has a high frequency of integration and it can infect nondividingcells, thus making it useful for delivery of genes into mammalian cells,for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has abroad host range for infectivity (Tratschin et al., 1984; Laughlin etal., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein byreference.

Retroviral Vectors

Retroviruses have promise as delivery vectors due to their ability tointegrate their genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding a polynucleotide of interest) is inserted into the viral genomein the place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson etal., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al.,1986; Potter et al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); byusing DEAE dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau etal., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved by cotransfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with amiRNA-198 therapy or polynucleotide encoding same. The tissue may bepart or separated from an organism. In certain embodiments, a tissue maycomprise, but is not limited to, adipocytes, alveolar, ameloblasts,axon, basal cells, blood (e.g., lymphocytes), blood vessel, bone, bonemarrow, brain, breast, cartilage, cervix, colon, cornea, embryonic,endometrium, endothelial, epithelial, esophagus, facia, fibroblast,follicular, ganglion cells, glial cells, goblet cells, kidney, liver,lung, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood,prostate, skin, skin, small intestine, spleen, stem cells, stomach,testes, and all cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g., a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art.

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials. An appropriate host can be determined byone of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Cell typesavailable for vector replication and/or expressioninclude, but are notlimited to, bacteria, such as E. coli (e.g., E. coli strain RR1, E. coliLE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coliW3110 (F, lambda, prototrophic, ATCC No. 273325), DH5α, JM109, and KC8,bacilli such as Bacillus subtilis; and other enterobacteriaceae such asSalmonella typhimurium, Serratia marcescens, various Pseudomonas specie,as well as a number of commercially available bacterial hosts such asSURE® Competent Cells and SOLOPACK® Gold Cells (STRATAGENE®, La Jolla).In certain embodiments, bacterial cells such as E. coli LE392 areparticularly contemplated as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents such as the miRNA-198 therapy andmay include one or more additional agents, wherein any of the agents aredissolved or dispersed in or provided with a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof a pharmaceutical composition that comprises at least one miRNA-198therapy and, in some embodiments, an additional active ingredient, willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The miRNA-198 therapy may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The miRNA-198 therapy may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include an miRNA-198therapy, one or more lipids, and an aqueous solvent. As used herein, theterm “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds are well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compoundswhich contain long-chain aliphatic hydrocarbons and their derivatives. Alipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof. Of course, compounds other than those specificallydescribed herein that are understood by one of skill in the art aslipids are also encompassed by the compositions and methods of thepresent invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the miRNA-198 therapy may be dispersed in asolution containing a lipid, dissolved with a lipid, emulsified with alipid, mixed with a lipid, combined with a lipid, covalently bonded to alipid, contained as a suspension in a lipid, contained or complexed witha micelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the miRNA-198 therapyis formulated to be administered via an alimentary route. Alimentaryroutes include all possible routes of administration in which thecomposition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

Parenteral Compositions and Formulations

In further embodiments, miRNA-198 therapy may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example, but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and5,399,363 (each specifically incorporated herein by reference in itsentirety)..

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compound maybe formulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

Combination Treatments

In certain aspects, the therapy of the invention may be combined withother agents that are effective in the treatment of hyperproliferativedisease, such as anti-cancer agents. An “anti-cancer” agent is capableof negatively affecting cancer in a subject, for example, by killingcancer cells, inducing apoptosis in cancer cells, reducing the growthrate of cancer cells, reducing the incidence or number of metastases,reducing tumor size, inhibiting tumor growth, reducing the blood supplyto a tumor or cancer cells, promoting an immune response against cancercells or a tumor, preventing or inhibiting the progression of cancer, orincreasing the lifespan of a subject with cancer. The compositions ofthe present invention are considered anti-cancer agents.

More generally, these other compositions or methods would be provided ina combined amount effective to kill or inhibit proliferation of thecancer cell. This process may involve contacting the cells withmiRNA-198, which may be referred to as the first agent, and the secondagent(s) or multiple factor(s) at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the expression construct and the other includes thesecond agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver, et al., 1992). In the context ofthe present invention, it is contemplated that the miRNA-198 therapycould be used similarly in conjunction with chemotherapeutic,radiotherapeutic, or immunotherapeutic intervention, in addition toother pro-apoptotic or cell cycle regulating agents, for example.

Alternatively, the inventive therapy may precede or follow the otheragent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one may contact the cell with both modalities withinabout 12-24 h of each other and, more preferably, within about 6-12 h ofeach other. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several d (2, 3, 4,5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

Various combinations may be employed, wherein inventive therapy is “A”and the secondary agent, such as radio- or chemotherapy, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the therapeutic agents of the present invention to apatient will follow general protocols for the administration ofchemotherapeutics, taking into account the toxicity, if any, of thecomposition. It is expected that the treatment cycles would be repeatedas necessary. It also is contemplated that various standard therapies,as well as surgical intervention, may be applied in combination with thedescribed hyperproliferative cell therapy.

Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, oxaliplatin,taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing. Any of thesetherapies may be used in combination with the invention.

There is no effective specific chemotherapeutics for pancreatic cancer.The way the chemotherapy is given depends on factors such as the typeand stage of the cancer being treated, and one of skill in the art wouldidentify which chemotherapy to use given the type and stage of thecancer, for example. Systemic chemotherapy with single-agent gemcitabineor a gemcitabine-based regimen still remains one of the standards ofcare for the treatment of patients with locally advanced and metastaticpancreatic cancer. A recent report showed that addition of Gemcitabineto radiation and 5-FU treatment after the surgery helped patient livelonger. For advanced, inoperable pancreatic cancer, patients withcombination of Gemcitabine and Cisplatin or oxaliplatin (Eloxatin)treatment survive longer than single drug treatment.

Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as 7-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with the inventive therapy. The general approach forcombined therapy is discussed below. Generally, the tumor cell must bearsome marker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155.

Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the miRNA-198. A variety of proteins are encompassedwithin the invention, some of which include inducers of apoposis and/orinhibitors of cell proliferation, for example.

Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abililties of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adehesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

Kits of the Invention

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, miRNA-198 and an additional agent, including anadditional anti-cancer agent, in specific embodiments, may be comprisedin a kit. The kits will thus comprise its contents in suitable containermeans.

The kits may comprise a suitably aliquoted miRNA-198 therapy, apharmaceutical carrier, such as a lipid, and including a liposome,and/or an additional agent. Compositions of the present invention,whether labeled or unlabeled, as may be used to prepare a standard curvefor a detection assay. The components of the kits may be packaged eitherin aqueous media or in lyophilized form. The container means of the kitswill generally include at least one vial, test tube, flask, bottle,syringe or other container means, into which a component may be placed,and preferably, suitably aliquoted. Where there are more than onecomponent in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the miRNA-198 therapy, lipid,additional agent, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection or blowmolded plastic containers into which the desired vials are retained.

Therapeutic kits of the present invention are kits that may comprise anmiRNA-198. Additional agents may include chemical compounds orpharmaceutically acceptable salts thereof, a protein, polypeptide,peptide, inhibitor, gene, polynucleotide, vector and/or other effector.Such kits may generally contain the compositions in a pharmaceuticallyacceptable formulation. The kit may have a single container means,and/or it may have distinct container means for each compound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The agent may also beformulated into a syringeable composition. In this case, the containermeans may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an affected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. The formulation may besuitable for systemic or local delivery.

In some embodiments, the components of the kit may be provided as driedpowder(s). When reagents and/or components are provided as a dry powder,the powder can be reconstituted by the addition of a suitable solvent.It is envisioned that the solvent may also be provided in anothercontainer means and may be sterile.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which theinhibitory formulation is placed, and preferably is suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of theultimate composition within or to the body of an animal. Such aninstrument may be a syringe, pipette, forceps, and/or any such medicallyapproved delivery vehicle.

In specific embodiments, the kit comprises an additional composition fortreatment of cancer, including a chemotherapeutic drug. The kit may betailored to include chemotherapeutic drugs suitable for the type ofcancer being treated. For example, kits may be formulated forindividuals with pancreatic cancer and may include in addition to themiRNA-198 one or more breast cancer drugs, such as Taxol, herceptin,tamoxifen, paclitaxel, gemcitabine, and so forth.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of certainembodiments and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1 MiR-198 is Downregulated in Patient Tumors, and its ExpressionCorrelates with Patient Survival

Profiling PC specimens has revealed a dysregulation of miRNA expressionwith respect to normal tissue or chronic pancreatitis (Bloomston et al.,2007; Zhang et al., 2009); however, the functional consequences of theseexpression changes have not been further dissected. miRNA expressionprofiling was applied to study the molecular mechanisms of MSLN-mediatedPC pathogenesis. In a previous study MSLN was overexpressed in MIA-PaCa2cells, which have low endogenous MSLN levels, and it was found that MSLNoverexpression led to increased migration, invasion, and proliferationboth in vitro and in vivo (Bharadwaj et al., 2008; Bharadwaj et al.,2011b). Here, the expression levels of 95 cancer-associated miRNAs inMSLN overexpressing cells (MIA-MSLN) cells and vector control cells(MIA-V) was analyzed by real-time RT-PCR. A global dysregulation ofmiRNA expression was found, with several miRNAs being either upregulated(for instance, miR-10b, miR-196a) or downregulated (for instance,miR-198, miR-200c, miR-155) following MSLN overexpression (FIG. 1A,Table 1). MiR-198 was the most significantly downregulated of all theexamined miRNAs, with a downregulation of ˜15-fold. In an embodiment ofthe invention, overexpression of MSLN initiates a chain of eventsculminating in repression of miR-198 in pancreatic tumors, with theresult being increased tumor growth and progression.

TABLE 1Primers for 95 cancer-associated miRNAs and U6 snRNA control included inmiRNA array shown in FIG. 2. miRNA miRBASE# miRNA sequence let-7-familyMIMAT0000062, ugagguaguagguuguauaguu, (SEQ ID NO. 2) MIMAT0000064,ugagguaguagguuguaugguu, (SEQ ID NO. 3) MIMAT0000065,agagguaguagguugcauagu, (SEQ ID NO. 4) MIMAT0000067ugagguaguagauuguauaguu (SEQ ID NO. 5) miR-7 MIMAT0000252Uggaagacuagugauuuuguug (SEQ ID NO. 6) miR-92 MIMAT0000092Uauugcacuugucccggccug (SEQ ID NO. 7) miR-93 MIMAT0000093Aaagugcuguucgugcagguag (SEQ ID NO. 8) miR-9-1 MIMAT0000441Ucuuugguuaucuagcuguauga (SEQ ID NO. 9) miR-101-1 MIMAT0000099Uacaguacugugauaacugaag (SEQ ID NO. 10) miR-103 M1MAT0000101Agcagcauuguacagggcuauga (SEQ ID NO. 11) miR-106a MIMAT0000103Aaaagugcuuacagugcagguagc (SEQ ID NO. 12) miR-106b M1MAT0000680Uaaagugcugacagugcagau (SEQ ID NO. 13) miR-107 MIMAT0000104Agcagcauuguacagggcuauca (SEQ ID NO. 14) miR-10b MIMAT0000254Uacccuguagaaccgaauuugu (SEQ ID NO. 15) miR-1-1 MIMAT0000416Uggaauguaaagaaguaugua (SEQ ID NO. 16) miR-122a MIMAT0000421Uggagugugacaaugguguuugu (SEQ ID NO. 17) miR-125a MIMAT0000443Ucccugagacccuuuaaccugug (SEQ ID NO. 18) miR-125b MIMAT0000423Ucccugagacccuaacuuguga (SEQ ID NO. 19) miR-126 MIMAT0000444Cauuauuacuuuugguacgcg (SEQ ID NO. 20) miR-128b MIMAT0000676Ucacagugaaccggucucuuuc (SEQ ID NO. 21) miR-132 MIMAT0000426Uaacagucuacagccauggucg (SEQ ID NO. 22) miR-133a MIMAT0000427Uugguccccuucaaccagcugu (SEQ ID NO. 23) miR-134 MIMAT0000447Ugugacugguugaccagaggg (SEQ ID NO. 24) miR-135b MIMAT0000758Uauggcuuuucauuccuaugug (SEQ ID NO. 25) miR-136 MIMAT0000448Acuccauuuguuuugaugaugga (SEQ ID NO. 26) miR-137 MIMAT0000429Uauugcuuaagaauacgcguag (SEQ ID NO. 27) miR 141 MIMAT0000431Agugguuuuacccuaugguag (SEQ ID NO. 28) miR-141 MIMAT0000432Uaacacugucugguaaagaugg (SEQ ID NO. 29) miR-142-3p MIMAT0000434Uguaguguuuccuacuuuaugga (SEQ ID NO. 30) miR-143 MIMAT0000435Ugagaugaagcacuguagcuca (SEQ ID NO. 31) miR-145 MIMAT0000437Guccaguuuucccaggaaucccuu (SEQ ID NO. 32) miR-146a MIMAT0000449Ugagaacugaauuccauggguu (SEQ ID NO. 33) miR-149 MIMAT0000450Ucuggcuccgugucuucacucc (SEQ ID NO. 34) miR-150 MIMAT0000451Ucucccaacccuuguaccagug (SEQ ID NO. 35) miR-151 MIMAT0000757Acuagacugaagcuccuugagg (SEQ ID NO. 36) miR-153 MIMAT0000439Uugcauagucacaaaaguga (SEQ ID NO. 37) miR-154 MIMAT0000452Uagguuauccguguugccuucg (SEQ ID NO. 38_ miR-155 MIMAT0000646Uuaaugcuaaucgugauagggg (SEQ ID NO. 39) miR-15a MIMAT0000068Uagcagcacauaaugguuugug (SEQ ID NO. 40) miR-15b MIMAT0000417Uagcagcacaucaugguuuaca (SEQ ID NO. 41) miR-16 MIMAT0000069Uagcagcacguaaauauuggcg (SEQ ID NO. 42) miR-17-3p MIMAT0000071Acugcagugaaggcacuugu (SEQ ID NO. 43) miR-17-5p MIMAT0000070Caaagugcuuacagugcagguagu (SEQ ID NO. 44) miR-181a MIMAT0000256Aacauucaacgcugucggugagu (SEQ ID NO. 45) miR-181b MIMAT0000257Aacauucauugcugucgguggg (SEQ ID NO. 46) miR-181c MIMAT0000258Aacauucaaccugucggugagu (SEQ ID NO. 47) miR-181d MIMAT0002821Aacauucauuguugucgguggguu (SEQ ID NO. 48) miR-183 MIMAT0000261Uauggcacugguagaauucacug (SEQ ID NO. 49) miR-185 MIMAT0000455Uggagagaaaggcaguuc (SEQ ID NO. 50) miR-296 MIMAT0000690Agggcccccccucaauccugu (SEQ ID NO. 51) miR-186 MIMAT0000456Caaagaauucuccuuuugggcuu (SEQ ID NO. 52) miR-188 MIMAT0000457Caucccuugcaugguggagggu (SEQ ID NO. 53) miR-18a MIMAT0000072Uaaggugcaucuagugcagaua (SEQ ID NO. 54) miR-190 MIMAT0000458Ugauauguuugauauauuaggu (SEQ ID NO. 55) miR-191 MIMAT0000440Caacggaaucccaaaagcagcu (SEQ ID NO. 56) miR-192 MIMAT0000222Cugaccuaugaauugacagcc (SEQ ID NO. 57) miR-194 MIMAT0000460Uguaacagcaacuccaugugga (SEQ ID NO. 58) miR-195 MIMAT0000461Uagcagcacagaaauauuggc (SEQ ID NO. 59) miR-196a MIMAT0000226Uagguaguuucauguuguugg (SEQ ID NO. 60) miR-197 MIMAT0000227Uucaccaccuucuccacccagc (SEQ ID NO. 61) miR-198 MIMAT0000228gguccagaggggagauagg (SEQ ID NO: l) miR-199a + b MIMAT0000231,cccaguguucagacuaccuguuc, (SEQ ID NO. 62) MIMAT0000263cccaguguuuagacuaucuguuc (SEQ ID NO. 63) miR-30b MIMAT0000420Uguaaacauccuacacucagcu (SEQ ID NO. 64) miR-19a + b MIMAT0000073,ugugcaaaucuaugcaaaacuga, (SEQ ID NO, 65) MIMAT0000074ugugcaaauccaugcaaaacuga (SEQ ID NO. 66) miR-95 MIMAT0000094Uucaacggguauuuauugagca (SEQ ID NO. 67) miR-20a MIMAT0000075Uaaagugcuuauagugcagguag (SEQ ID NO. 68) miR-200a MIMAT0000682Uaacacugucugguaacgaugu (SEQ ID NO. 69) miR-200b MIMAT0000318Uaauacugccugguaaugaugac (SEQ ID NO. 70) miR-200c MIMAT0000617Uaauacugccggguaaugaugg (SEQ ID NO. 71) miR-202 MIMAT0002811Agagguauagggcaugggaaaa (SEQ ID NO. 72) miR-203 MIMAT0000264Gugaaauguuuaggaccacuag (SEQ ID NO. 73) miR-204 MIMAT0000265Uucccuuugucauccuaugccu (SKQ ID NO. 74) miR-205 MIMAT0000266Uccuucauuccaccggagucug (SEQ ID NO. 75) miR-206 MIMAT0000462Uggaauguaaggaagugugugg (SEQ ID NO. 76) miR-21 MIMAT0000076Uagcuuaucagacugauguuga (SEQ ID NO. 77) miR-210 MIMAT0000267Cugugcgugugacagcggcuga (SEQ ID NO. 78) miR-214 MIMAT0000271Acagcaggcacagacaggcag (SEQ ID NO. 79) miR-215 MIMAT0000272Auguccuuugaauugacugac (SEQ ID NO. 80) miR-372 MIMAT0000724Aaagugcugcgacauuugagcgu (SEQ ID NO. 81) miR-373 MIMAT0000726Gaagugcuucgauuuuggggugu (SEQ ID NO. 82) miR-22 MIMAT0000077Aagcugccaguugaagaacugu (SEQ ID NO. 83) miR-488 MIMAT0002804Cccagauaauggcacucucaa (SEQ ID NO. 84) miR-221 MIMAT0000278Agcuacauugucugcuggguuuc (SEQ ID NO. 85) miR-222 MIMAT0000279Agcuacaucuggcuacugggucuc (SEQ ID NO. 86) miR-223 MIMAT0000280Ugucaguuugucaaauacccc (SEQ ID NO. 87) miR-224 MIMAT0000281Caagucacuagugguuccguuua (SEQ ID NO. 88) miR-23a MIMAT0000078Aucacauugccagggauuucc (SEQ ID NO. 89) miR-24 MIMAT0000080Uggcucaguucagcaggaacag (SEQ ID NO. 90) miR-25 MIMAT0000081Cauugcacuugucucggucuga (SEQ ID NO. 91) miR-26a MIMAT0000082Uucaaguaauccaggauaggc (SEQ ID NO. 92) miR-26b MIMAT0000083Uucaaguaauucaggauagguu (SEQ ID NO. 93) miR-27a + b MIMAT0000084uucacaguggcuaaguuccgc, (SEQ ID NO. 94) MIMAT0000419uucacaguggcuaaguucugc (SEQ ID NO. 95) miR-30c MIMAT0000244Uguaaacauccuacacucucagc (SEQ ID NO. 96) miR- MIMAT0000086,uagcaccaucugaaaucgguu, (SEQ ID NO. 97) 29a + 30b + 30c MIMAT0000100,uagcaccauuugaaaucaguguu, (SEQ ID NO. 98) MIMAT0000681uagcaccauuugaaaucggu (SEQ ID NO. 99) miR-30a-3p MIMAT0000088Cuuucagucggauguuugcagc (SEQ ID NO. 100) miR-30a-5p MIMAT0000087Uguaaacauccucgacuggaag (SEQ ID NO. 101) U6 snRNA NCBI: X07425.1Caccacguuuauacgccggug (SEQ ID NO. 102)

To determine the clinical relevance of miR-198 expression in PC miR-198expression in 37 PC patients was examined, and it was found that miR-198was downregulated in 78.4% of tumors compared to adjacent normal tissues(FIG. 1B), with downregulation ranging from 2-142 fold, and an averagedownregulation of 10 fold (Table 2). The cohort of patients wereclassified into two groups based on miR-198 tumor levels: miR-198-Lowand miR-198-High. All patients in the miR-198-Low group (n=26), had amiR-198 level of <1*10⁻³ (relative to U6 control), with an averagerelative miR-198 level of 0.0004; all patients in miR-198-High group hada minimum of a ten-fold higher level of miR-198, with a level of >1*10⁻²and an average relative miR-198 of 0.08, a greater than 100 foldincrease over the miR-198-Low group (p<0.0005).

TABLE 2 Fold changes of network factors in tumor tissues compared tomatched normal tissues for PC patients. Patient # OCT-2 VCP PBX-1 MSLNMIR-198 FSTL1 miR- 1 13.90 4.42 2.91 1.87 −4.44 −1.43 198 2 5.15 5.756.08 7.61 −2.89 −1.39 low 3 6.96 7.46 3.32 6.25 −6.80 −3.86 4 41.4417.76 51.51 30.12 −42.44 −45.95 5 −5.88 6 −2.23 7 2.20 10.44 8.46 1.92−4.71 −4.17 8 15.93 43.67 56.49 30.55 −24.03 −10.26 9 7.02 11.91 28.2113.71 −10.16 −7.82

Patient survival in the two groups was examined. As measured by Log-ranktest, patient survival was significantly increased in the miR-198-Highgroup compared to the low group, (p=0.0002, Chi Square=14.12 df=1), witha hazard ratio of 5.815 (95% Cl of ratio 2.321 to 14.57) (FIG. 1C). Themedian survival of the miR-198-Low patients was ˜15.5 months, with only11% of patients still alive after ˜40 months. Conversely, miR-198-Highpatients who had died had a median survival time of 35.75 months, with80% of miR-198-High patients were still alive at 40 months, and 64%still alive after ˜60 months. These results clearly implicate theclinical significance of miR-198 being an important prognostic factor inPC.

Example 2 An Interactome of Tumorigenic Factors Interconnected ThroughmiR-198 Serves as a Prognostic Indicator of PC

MiR-198 is an intronic miRNA, located in the 3′UTR of the gene for humanfollistatin related protein (FSTL-1) (Cullen, 2004; Rodriguez et al.,2004). While most intronic miRNAs tend to be under the control of thesame promoter as their host gene, the relationship between FSTL1 andmiR-198 had not yet been established. Therefore, the FSTL1 mRNA levelsin the matched tissues was examined, and a close correlation withmiR-198 expression (p<0.05, R²=0.95) was found. The miR-198-Low grouphad an average relative FSTL1 mRNA level (normalized to β-actin) of0.01, while it was ten-fold higher in the miR-198-High group, at 0.10(FIG. 9A).

In silico analyses was performed on the FSTL-1/miR-198 promoter todetermine potential binding sites for repressive transcription factorsthat could be downregulating miR-198 expression in tumors (Table 3). Thepotential involvement of several transcription factors including OCT-2,ZEB-1 and KLF4 (data not shown) was also studied. Several octamer motifbinding sites for the repressive POU domain binding factor OCT-2(consensus sequence ATGCAAAT) were identified. While OCT-2 expressionhad never been studied in PC, here it was found that OCT-2 was expressedin the pancreas, and was upregulated in ˜81% of patient tumors (FIG.9A). OCT-2 mRNA expression, while low in normal tissues, was upregulated˜6 fold on average in matched patient tumors (Table 2). When the OCT-2expression was examined in the two cohorts of patients segregated bymiR-198 levels, a clear distinction was found between the two groupswith respect to OCT-2 levels (FIG. 9A). The average OCT-2 tumorexpression level was 3.2*10⁻⁵ in the miR-198-High group (relative toβ-actin), while this was upregulated to an average 4.4*10⁻³ in themiR-198-Low group, a >100-fold change in relative expression of OCT-2 inthe tumor tissues between the two groups (p=0.25, 95% CI=−0.003391 to0.01214).

TABLE 3 Conserved Transcription Factor Binding Sites in FSTL1/miR- 198promoter region. Number of OCT-2 binding sites in bold. TranscriptionNumber of Sites in Factor Name miR-198 promoter AIRE 1 AREB6 2 BACH2 1BARBIE 1 CETS16B 1 CP2 1 CREB 2 CREBATF 1 CREL 1 DR3 1 E12 1 E2F 1 E2F11 E2F1DP1 1 E2F1DP2 1 E2F4DP2 1 E47 2 ELF1 2 EVI1 1 FOXJ2 1 FREAC3 1GABP 1 GCM 1 HAND1E47 2 HEB 1 HES1 1 HFH3 1 HFH8 1 HNF3ALPHA 1 HNF3B 1IK3 1 KLF4 6 LDSPOLYA 1 MAZR 1 MMEF2 1 MTATA 1 MYOD 2 NFKAPPAB65 1 NKX251 NRF2 1 OCT1/OCT2 3 OSF2 2 PBX-1 1 POUF1 1 RFX1 2 TAL1 1 TATA 1 TCF1P 3TITF1 2 USF 1 XFD1 1 XFD2 1 XFD3 1 ZEB1 17

MSLN was upregulated in 77% of patient tumors, with an average of 8.6fold increase in expression in tumors over normal tissues (Table 2).There was a significant difference in the average relative MSLN levelsin patient tumors between the miR-198-Low (1.8×10⁻²) and miR-198-Highgroups (8.5×10⁻⁴) (p<0.05) (FIG. 9A).

MiRNA target prediction software was used to find potential miR-198targets through which miR-198 might be modulating tumorigenesis in PCcells. A microarray was used to screen targets for changes in expressionwithin the miR-198-High and miR-198-Low groups and selected twocandidates that had a differential expression in response to miR-198levels, PBX-1 and VCP. A potential miR-198 binding site was identifiedwithin the 3′UTR of PBX-1 (FIG. 10A). This binding site isevolutionarily conserved in 22 of 23 species examined (FIG. 10C). Inaddition to its direct effects on tumorigenicity, PBX-1 is also aregulator of VCP, and increased expression of the PBX-1-VCP axis hasbeen associated with cancer metastasis, increased cell survival, andpoor prognosis of patients with PC (Qiu et al., 2007). It was also foundthat VCP also has a binding site for miR-198 within its 3′UTR (FIG.10B), which is conserved in a majority of species (FIG. 10D). Both PBX-1and VCP were upregulated in a majority of patient tumors (81.3% and87.1%, respectively), with an average upregulation of ˜9 and ˜6 fold,respectively. A clear distinction was observed between the miR-198-Lowand High groups, with an average relative PBX-1 and VCP level ˜10 foldhigher in the miR-198-Low group compared to the miR-198-High group(PBX-1: 0.04 and 0.003, p<0.05, and VCP: 0.12 and 0.013, respectively.p<0.05) (Table 2, FIG. 9A).

If the factors in this network are linked through miR-198, then thechanges in their expression of all of the factors would correlate witheach other. FIG. 1D shows the linear regression analysis for the foldchanges in expression of all these factors between the tumor and normalmatched samples in relation to miR-198. FSTL1 correlates with miR-198(p<0.0005, R²=0.95), while there is a negative correlation betweenmiR-198 and OCT-2 (p<0.0005, R²=0.79), MSLN (p<0.0005, R²=0.92), PBX-1(p<0.0005, R²=0.63), and VCP (p<0.05, R²=0.14) in patient tumor tissues.All these factors also correlate positively with each other andnegatively with miR-198 and FSTL1 (FIG. 9B), indicating that the changesin their expression may be dependent on each other as members of thisnetwork.

The complex interactome between the factors was further representedusing the five-order Venn diagrams depicted in FIG. 1, E-G. The foldchanges of each factor was examined between tumor and adjacent normaltissues and the percentage of times upregulation occurred from normal totumor was calculated for MSLN, OCT-2, PBX-1, or VCP individually, andhow often the upregulation in each was accompanied by an upregulation inthe other factors as well as downregulation of miR-198. To reducecomplexity, FSTL1 was not included in this graphical representation, asits close correlation with miR-198 expression makes them interchangeablein this analysis. As shown in FIG. 1E, for the total (n=37) samples ofpatient tissues, a downregulation in miR-198 was accompanied by asimultaneous upregulation in all other network members (MSLN, OCT-2,PBX-1, and VCP) in 71% of tumors. When the same analysis was performedfor either the miR-198-Low or miR-198-High groups separately, a veryclear change in the molecular makeup of this interactome was observed.As shown in FIG. 1F, a simultaneous upregulation of MSLN, OCT-2, PBX-1,and VCP and downregulation of miR-198 occurs in 91.7% of the patientswith the worst survival prognosis. This percentage is the same for allthe other possible permutations of interactions between the differentfactors, indicating a very close association between their expressionchanges in this subset of patients. An intriguing result is shown inFIG. 1G. In the miR-198-High group, the cohort with the highest survivalprognosis none (0%) of the patients shows a simultaneous modulation ofthe five factors in the interactome. This also extends to most of theother potential combinations in expression changes.

These results indicate that this interactive tumor signature network isvery tightly correlated in PC patients with the worst prognosis, whilethose with the best prognosis have a global disruption in the networkexpression pattern; these tumors have a different functional makeupeither due to or resulting in an elevated level of miR-198 and adecreased level of OCT-2, MSLN, PBX-1, and VCP.

Example 3 MSLN-Mediated NF-kB Activation Represses the miR-198 PromoterThrough OCT-2 Induction

Having demonstrated that MSLN overexpression resulted in miR-198repression (FIG. 1A), the mechanism through which MSLN was repressingmiR-198 was then determined. The findings were first confirmed in adifferent cell line to rule out cell-line specific effects byoverexpressing MSLN in HPDE control cells. This resulted in a similar10-fold reduction in miR-198 expression p<0.05 (FIG. 2A). Conversely,transfection of MSLN-specific shRNAs in MSLN-overexpressing cellsrestored miR-198 expression ˜12 fold in MIA-MSLN cells, furtherindicating that MSLN was responsible for the observed miR-198downregulation (p<0.05) (FIG. 2B). That is, silencing MSLN restoresmiR-198 expression.

To verify that an in vitro system would recapitulate the results of theclinical data, miR-198 and MSLN expression were examined in a panel ofPC cell lines (FIG. 2C) and it was found that when MSLN expression washigh, miR-198 was low, which parallels observations in the patienttumors. That is, MSLN mRNA levels correlate negatively with miR-198levels in human PC cells. Fold change was calculated relative to PC cellwith lowest expression of each factor. Compared to HPDE cells and normaltissues, miR-198 expression is downregulated in majority of PC celllines (FIG. 2C). Cell lines with low MSLN levels, such as MIA-PaCa2 andPanc-28, had the highest miR-198 levels among the PC cells, ranging from100-700 fold greater miR-198 expression than AsPC-1 cells (which havethe highest MSLN levels). Conversely, cell lines with relatively highMSLN mRNA levels (>50-fold higher expression than HPDE), had the lowestlevels of miR-198 (FIG. 2C), had a pronounced negative relationshipbetween MSLN expression at the protein level and miR-198 expression(FIG. 3A).

FSTL1 expression was also closely correlated with miR-198 expression(p<0.005) in the cell line panel (FIG. 2D; p<0.001, R2=0.87). Inaddition, FSTL1 mRNA levels were reduced by ˜80% in MIA-MSLN cellscompared to MIA-V cells following forced MSLN expression (p<0.005) (FIG.2E). These results confirm that expression of miR-198 and its host geneFSTL1 are in fact linked, and that MSLN overexpression leads to aconcomitant reduction in expression of both.

Constitutive NF-κB activation occurs in PC cells in response to MSLNoverexpression (Bharadwaj et al., 2011a). OCT-2 transcription is inducedby NF-κB activation in B-cells (Liu et al., 1996). It was previouslyreported that MSLN constitutively activates NF-κB in PC cells (Bharadwajet al., 2011a; Bharadwaj et al., 2011b). No to be limited, theMSLN-mediated NF-κB activation may be linked to OCT-2overexpression/miR-198 downregulation. Several binding sites wereidentified for the NF-κB-inducible repressive transcription factor OCT-2in the FSTL1/miR-198 promoter, as well as a close association betweenOCT-2, MSLN, and miR-198 (FIG. 1D). Taken together these findings led tothe nonbinding postulate that MSLN-mediated NF-κB activation andsubsequent OCT-2 induction might provide a link between MSLNoverexpression and miR-198 repression. To test this, MIA-MSLN cells weretreated with the NF-κB inhibitor Wedelolactone, and a ˜17-fold increasein miR-198 expression was observed when compared to untreated controls(FIG. 2F). That is, wedelolactone treatment restores miR-198 expressionin MIA-MSLN cells to pre-MSLN levels. Forced MSLN expression led to astrong induction in OCT-2 expression. While MIA cells had very lowendogenous OCT-2 levels, MIA-MSLN cells had high levels of OCT-2 (FIG.11A). Treatment of MIA-MSLN cells with Wedelolactone led to an almostcomplete block in OCT-2 expression at both the mRNA and protein levels,indicating that MSLN-mediated NF-κB activation was in fact responsiblefor the induction of OCT-2 (FIG. 2G). That is, wedelolactone treatmentblocks OCT-2 induction in MIA-MSLN cells. These results were alsoconfirmed in endogenously high MSLN cell lines (FIG. 11B).

OCT-2 can function as a repressor or an activator depending on thecell-specific context (Azuara-Liceaga et al., 2004; Dawson et al., 1994;Liu et al., 1996). OCT-2-specific shRNAs were used to knock down OCT-2expression and to determine whether it was acting as a repressor formiR-198. OCT-2 knockdown results in a 12-fold upregulation of miR-198expression after 72 h, restoring miR-198 levels close to pre-MSLNexpression levels (FIG. 2H). These results demonstrate that OCT-2overexpression resulting from MSLN-mediated NF-κB activation stronglyrepresses miR-198, and that blocking either NF-κB or OCT-2 in thispathway can effectively restore miR-198 expression.

Example 4 MiR-198 is the Central Link Between Upstream RegulatoryFactors MSLN and OCT-2 and the Closely Correlated Downstream PBX-1/VCPTumorigenic Axis

The above studies established correlations between the various factorsin the interactome. These relationships were then verified in vitro sothat the mechanisms through which these molecules interacted could befurther elucidated. A series of western blots were performed in order todemonstrate the link between upstream MSLN and OCT-2 regulators and thedownstream PBX-1/VCP axis (FIG. 3A-C). As shown in FIG. 3A, MSLNexpression correlated closely with PBX-1 expression (p<0.05) andinversely with miR-198 expression in a majority of PC cell lines. Thesame correlation was confirmed at the mRNA level (FIGS. 10E and 10F).

OCT-2 and PBX-1 expression was next examined in a PC cell line panel.HPDE cells had almost undetectable OCT-2 expression levels, while themajority of PC cell lines had a low level of OCT-2 expression. This isthe first report of OCT-2 expression in PC cell lines. Additionally,OCT-2 expression correlated closely with both MSLN expression and PBX-1expression, with markedly higher OCT-2 expression in most cells with thehighest MSLN and PBX-1 levels and low miR-198 levels (p<0.05) (FIG. 3B).Lastly, FIG. 3C demonstrates the correlation between OCT-2 and VCP,tying together the last of the factors in the interactome in the PC cellline panel, thereby confirming that the results observed from clinicalsamples at the mRNA level translate to protein expression with afunctional effect.

Both PBX-1 and VCP are predicted targets of miR-198, and thereforedownstream effectors of the network. To examine this relationship,miR-198 was stably overexpressed in MIA-MSLN cells and AsPC1 cells (FIG.12). Overexpressing miR-198 in MIA-MSLN or AsPC1 cells led to a decreasein PBX-1 mRNA (FIG. 10G, FIG. 10H) and protein expression (FIG. 3D),making them almost undetectable. A similar result was observed for VCPfollowing miR-198 reconstitution in MIA-MSLN cells; VCP proteinexpression in MIA-MSLN and AsPC1 cells is reduced following miR-198overexpression in both cell lines (FIG. 3E and FIG. 10I), although abasal level of expression is still detectable. At the mRNA level,MIA-MSLN cells had a consistent in VCP mRNA levels following miR-198overexpression (FIGS. 10I and J). These results were tied to the rest ofthe interactome using either Wedelolactone inhibition of NF-κB (FIG. 3F)or shRNAs against OCT-2 (not shown), both of which led to thedownregulation of PBX-1 expression.

Luciferase reporters and site-directed mutagenesis were used to eitherdelete the entire miR-198 binding site or to insert three pointmutations within the miR-198 seed region of both PBX-1 and VCP (FIGS.10K and L). Dual-luciferase assays showed a 65% reduction in PBX-1luciferase expression following miR-198 transfection, which wasabolished when the target site for miR-198 was either deleted or mutated(FIG. 3G), indicating a direct targeting of miR-198 to the 3′UTR ofPBX-1. In the case of VCP, a ˜70% decrease in luciferase activity(p<0.05) was partially restored when the seed region was mutated (FIG.3H).

Example 5 MiR-198 Reciprocally Regulates MSLN Expression by Binding toTarget Sites within the MSLN Coding Region (CDS)

Interestingly, miR-198 overexpression in MIA-MSLN cells was observed tolead to an almost complete reduction of MSLN expression at the proteinlevel (FIG. 4A), with a partial decrease in the mRNA expression levels(FIG. 4B), indicating a mechanism of post-transcriptional regulation.This suggestes that a reciprocal regulatory loop and feed-forwardconstitutive activation pathway may exist within the interactome, withMSLN modulating miR-198 through the NF-κB/OCT-2 axis and miR-198directly regulating MSLN expression.

No putative miR-198 targets were found in the 3′UTR of MSLN using targetprediction software. However, while miRNAs typically act on the 3′UTRsof transcripts, recent evidence suggests that miRNAs also act on the5′UTRs (Lytle et al., 2007) and even within the CDS of genes (Tay etal., 2008). RNA22 was used to search for potential miR-198 binding siteswithin the CDS of MSLN, and three potential binding sites for miR-198were found (FIG. 13A). Site-directed mutagenesis was used to alter thenucleotide sequence of the miR-198 binding sites without altering theamino acid composition of the MSLN protein (FIG. 13B), andco-transfected WT or mutant plasmids was transfected along with miR-198or control precursors into COS-7 cells, chosen because they haveundetectable endogenous levels of both MSLN and miR-198 (FIG. 13C).MiR-198 transfection abolished expression of the WT MSLN protein (FIG.4C). Mutating the three binding sites resulted in differentialregulation of MSLN by miR-198. When site 1 was mutated, there was almostno noticeable recovery of MSLN expression. However, mutating site 2resulted in an almost complete recovery of protein expression, with theexpression of the site 3 mutant falling in between the two. Doublemutants of sites 1+3 or 1+2 resulted in no noticeable improvement inexpression than their single counterparts. However, the site 2+3 doublemutant resulted in complete restoration of MSLN expression (FIG. 4C).This indicates that at least two miR-198 binding sites cooperate toblock MSLN protein expression.

MiR-198 targeting of the MSLN CDS regions was further validated byconstructing luciferase reporter constructs containing 600 or 400 bpportions of the MSLN CDS with WT or mutated seed regions (FIG. 13D).Firefly luciferase expression was significantly decreased in two of theWT constructs in the presence of miR-198 (almost completely for site 2,˜70% for site 3, p<0.05), and was restored to control levels in themutant constructs (FIG. 4D). While a slight reduction was also observedfor the site 1 constructs, it was not statistically significant.Mutating the miR-198 binding site failed to recover luciferaseexpression any further in this construct. RNA22 was used to examine theMSLN gene in several other species, including rats, mice, and monkeys,and it was found that all the species examined had 2 or 3 predictedmiR-198 binding sites within the MSLN coding region, despite the overalllack of homology of the MSLN protein with the rodent species (data notshown). Between Homo sapiens and Macaca mulatta, there is a 92% homologybetween the MSLN proteins. In this case, two of the three sites matchidentically between the two species: Site 2, which is located atposition 916-937 in the human MSLN CDS, is conserved at position 745-766in the monkey CDS; Site 3, which is located at position 511-532 in thehuman MSLN CDS, is conserved at position 344-365 in the monkey CDS. Bothof these binding sites correspond to the same region in both speciesbased on sequence homology. These results indicate the importance ofmiR-198 targeting of MSLN in that these sites have been evolutionarilyconserved. Taken together, the results show a synergistic effect betweenat least two sites in the MSLN coding region through which miR-198 canmodulate its expression, and that these sites are evolutionarilyconserved, underscoring the importance of this regulatory interaction.

Example 6 Reconstitution of miR-198 Reduces Tumorigenic Functions ofMSLN-Overexpressing Cells In Vitro

After demonstrating that miR-198 regulated expression of MSLN, PBX-1,and VCP in PC cells, the functional relevance of miR-198 modulation wasexamined and functional assays were performed to examine the effects ofmiR-198 expression on the tumorigenicity of MSLN-overexpressing PCcells. Transient transfection of miR-198 precursors in MIA-MSLN cellsled to a significant (p<0.05) decrease in proliferation, and migrationof MIA-MSLN cells (FIG. 14A-C). These results were validated byexamining the tumorigenic potential of the two lines of miR-198overexpressing stable cells (MIA-MSLN and AsPC1). After 48 h, migrationwas reduced by ˜40% in MIA-MSLN cells (p<0.05) (FIG. 5A), and by 33%(p<0.05) in AsPC1 cells (FIG. 14D). AsPC1-miR-198 cells hadsignificantly lower proliferation rates than AsPC1-miR-Ctrl cells (FIG.5B), as did MIA-V and MIA-MSLN-miR-198 cells, which both hadapproximately 3 fold lower proliferation rates than MIA-MSLN andMIA-MSLN-miR-Ctrl cells after 5 days (p<0.05) (FIG. 14E). Cell invasionwas measured across a Matrigel matrix, and observed a similar reductionin migration capacity following miR-198 overexpression (FIG. 5C, FIG.14F). Monolayer wound healing assays on both MIA-MSLN (FIG. 5D) andAsPC1 cells (FIG. 14G) show a reduced wound closure rate when miR-198was overexpressed in all the different serum conditions tested.

One of the additional hallmarks of MSLN overexpression is that it canconfer onto cells the ability for anchorage independent growth in softagar (Uehara et al., 2008). miR-198 overexpression resulted in a loss ofthis characteristic. MIA-MSLN cells were able to effectively establishcolonies in soft agar after 15 days, while MIA-MSLN-miR-198 plates hadvery few colonies. ImageJ software was used to analyze the density ofcolonies appearing on plates, and showed a reduction in mean thresholdintensity of almost 20 fold (p<0.05) (FIG. 5E, FIG. 14H).

The role of miR-198 in vitro was further examined using an antisenseinhibitor of miR-198 (Zip-198) (FIG. 12). Blocking miR-198 in MIA-Vcells (MIA-V-Zip-198) led to a significant increase in bothproliferation (FIG. 5F) and migration (p<0.05) (FIG. 5G), a result whichconfirmed in HPDE cells FIG. 14I).

It was previously found that MSLN overexpression results in increasedproliferation through activation of Stat3 (Bharadwaj et al., 2008) andincreased autocrine stimulation through IL6/sIL6R trans-signaling(Bharadwaj et al., 2011b). At the same time, PBX-1 has been shown to actas a transcription factor in cooperation with homeobox proteins toregulate proliferation and differentiation of both normal cells andcancer cells (Lu et al., 1995; Qiu et al., 2007). PBX-1 has also beenimplicated in metastasis and invasion (Asai et al., 2002), like MSLN (Liet al., 2008). In order to separate the effects of MSLN signaling fromthose of PBX-1 in PC cells, PBX-1 was modulated via overexpression orshRNA blocking in respective cell lines, which resulted in astatistically significant yet modest change in proliferation (FIG. 5H,FIG. 14J) when compared to the effects of MSLN modulation (p<0.05) (FIG.5B), thereby indicating that the MSLN/IL6/Stat3 axis plays more of arole in MSLN-mediated proliferation changes than the MSLN/PBX-1 axisdoes, although this relationship still contributes to the overalltumorigenesis of PC cells.

On the other hand, overexpression of PBX-1 in MIA-V or MIA-MSLN-miR-198cells results in increased migration resembling that observed followingMSLN overexpression in MIA-MSLN cells (p<0.05) (FIG. 5I). Conversely,when PBX-1 is blocked with shRNAs in MIA-MSLN cells, the migration ofthose cells drops to MIA-V levels. This indicates that PBX-1 isprimarily responsible for the increased invasiveness that resultsfollowing MSLN overexpression.

Example 7 MiR-198 Antagonizes the Pro-Survival Effects of MSLN in PCCells

It was previously reported that MSLN overexpression confers resistanceto TNF-α-induced apoptosis (Bharadwaj et al., 2011a). To test whethermiR-198 mediated modulation of MSLN could reverse this acquiredresistance, which would have strong clinical implications, a TUNEL assaywas performed in MSLN high cells following TNF-α treatment. After TNF-αtreatment, only ˜11% of control MIA-MSLN and ˜1.2% of AsPC1 cells wereundergoing apoptosis as measured by TUNEL. On the other hand,MIA-MSLN-miR-198 and AsPC1-miR-198 cells exposed to the same conditionsshow a dramatic increase in the number of cells undergoing apoptosis,of >60% and >90% over controls, respectively (FIG. 6A). Conversely,blocking miR-198 expression results in a significant and dramatic dropin the number of apoptotic cells, down to ˜19% in MIA-V-Zip-198 cells(FIG. 6B). Further, overexpressing miR-198 in MIA-V cells had a smallereffect in further increasing apoptosis from ˜68% to ˜75%, consistentwith the effects seen on proliferation (FIG. 5H). Apoptosis inductionwas confirmed through a western blot for caspase 3 activation. Thelevels of uncleaved caspase 3 decrease in miR-198 high cells (FIG. 6C).These results indicate that miR-198 can antagonize the cell survivaleffects conferred by MSLN overexpression and its subsequent modulationof downstream targets, implying that miR-198 replacement therapy couldpotentially reduce MSLN-induced chemotherapeutic resistance.

Example 8 MiR-198 Overexpression Reduces Tumor Growth and MetastaticSpread In Vivo

Mouse xenograft models were used to study the effects ofmiR-198-mediated modulation of the interactome. It was previouslyreported that MIA-MSLN cells show a dramatic increase in tumor volumecompared to MIA-V cells (Li et al., 2008). In accordance with thoseresults, it was found that altering the expression levels of the membersof the interactome through miR-198 overexpression resulted in areduction in tumor volume in two separate mouse models. In asubcutaneous (s.c.) model, 9/9 control mice developed large tumors withrapid onset (measurable at 7 days post injection), with an average tumorvolume of 2000 mm³ by time of sacrifice at 25 days post injection (FIG.7A). In contrast, 6/9 mice injected with miR-198 overexpressing cellsshowed no signs of tumor development throughout the course of the study,remaining tumor-free for 45 days. The three mice that presented smalltumors did not show measurable tumors until day 35 post injection, andby day 45 the tumors had grown to an average volume of only 37 mm³, ahighly significant difference compared to the control cell injected mice(p<0.0005) (FIG. 7A). Representative images are shown in FIG. 7B.

Mice injected orthotopically showed a similar phenotype. All miceinjected with MIA-MSLN-miR-Ctrl cells developed primary tumors with anaverage weight of 0.6 g, ˜10 fold greater than primary tumors in miceinjected with MIA-MSLN-miR-198 cells, (average weight of 0.06 g)(p<0.0005) (FIGS. 7, C and D). Control mice all had observable jaundice,weight loss, and abdominal ascitic fluid, while none of the miceinjected with high miR-198 cells had these symptoms. GFP expressioncassettes in stable cells allowed us to visualize the tumor spread inthese mice (FIG. 7E). The control mice all presented with liver, spleen,and intestinal metastasis, peritoneal dissemination, and local tumorspread throughout the body cavity. The MIA-MSLN-miR-198 injected mice,on the other hand, had decreased metastases in addition to the decreasedprimary tumor growth. Four of the eight mice had no metastases; one didnot develop any tumor; the remaining 3 mice showed limited tumor spread.

The expression levels were confirmed of the factors in the proposedinteractome in the mouse tumor tissues and found that miR-198overexpression in the smaller, less aggressive MIA-MSLN-miR-198 tumorswas accompanied by a significant decrease in expression of the otherfactors in the network as compared to their levels in the control tumors(p<0.05) (FIG. 7F). These results indicate that PC tumor progression issignificantly dampened in vivo following miR-198 reconstitution, likelythrough modulation of the molecular makeup of the tumors with regards tothe interactome.

Example 9 Materials and Methods of Examples 1-8

Cell Culture

Human PC cell lines used in this study were purchased from the ATCC andwere authenticated by DNA fingerprinting at the University of Texas MDAnderson Cancer Center Characterized Cell Line Core. HPDE cells wereprovided from the Ontario Cancer Institute, Canada. All cells werecultured as previously described (Bharadwaj et al., 2008; Li et al.,2008; Li et al., 2007).

Patient Tissue Collection

Human pancreatic adenocarcinoma specimens and adjacent normal tissueswere collected from patients who underwent surgery according to anapproved human IRB protocol (H-16215) at BCM (Houston, Tex.) and storedat the Elkins Pancreas Center tissue bank.

Subcutaneous and Orthotopic Pancreatic Cancer Mouse Models

All animal procedures were conducted under the guidelines approved bythe Institutional Animal Care and Use Committee (IACUC). Cells (3×10⁶)were inoculated into the right flank (s.c. model) or into the pancreas(orthotopic model) of 5 to 6-week-old male nude mice (NCI-Charles River)as previously described (Li et al., 2007). Tumor size was measured usinga digital caliper and tumor volume was determined with the followingformula: tumor volume (mm³)=[length (mm)]×[width (mm)]2×0.52. For theorthotopic model, after 4 weeks all surviving mice were euthanized andevaluated macroscopically for the presence of orthotopic tumor andmetastases in the abdominal cavity. Tumor spread was further visualizedusing a fluorescent filter to view GFP expression. The tumor noduleswere explanted, counted, and weighed. Tumor tissues were stored in RNAseLater solution (Ambion) in −80° for subsequent RNA/miRNA isolation andanalysis.

Statistical Analysis

Statistical analysis was performed using the Student's t-test (paired,2-tailed). Statistical significance was defined as p<0.05 (denoted by*). Survival data was analyzed using Log-rank test and the data wasplotted using the Kaplan-Meier method. Patients still living werecensored from the results. Data are presented as mean±SD. Linearregression and correlation analyses were performed using GraphPad Prism(GraphPad Software).

Immunoblotting

Western blots were performed as previously described (Li et al., 2008).Membranes were probed with: anti-MSLN monoclonal antibody (1:1000)(Abcam); PBX-1 polyclonal antibody (1:1000) and Caspase 3 antibody(1:1000) (Cell Signaling); OCT-2 polyclonal antibody (1:400), and VCPpolyclonal antibody (1:200) (Santa Cruz Biotechnology); or anti-β-actin,Tubulin, LaminA or GAPDH antibodies (1:10,000) (Sigma Aldrich).Secondary incubation was performed with horseradish peroxidase-linkedsecondary antibody (1:2,000) (anti-mouse, anti-goat or anti-rabbit)(Cell Signaling).

Cells were lysed in RIPA buffer (Invitogen) and protease inhibitorcocktail for 30 min in ice. Cell lysates were then collected aftercentrifugation at 12,000 rpm for 5 min at 4° C. Total proteinconcentration was quantified using BCA Protein Assay Reagent system(Thermo Scientific). Sixty micrograms of lysate protein was loaded on a10% SDS-PAGE gel, and total cellular protein was separated and thentransblotted overnight at 4° C. onto Hybond-P PVDF membrane (AmershamBiosciences). The membranes were probed with anti-MSLN monoclonalantibody (Abcam) (1:1,000), PBX-1 polyclonal antibody (Cell Signaling)(1:1,000), ZEB1 polyclonal antibody (1:1,000), cyclin T1 antibody(Baylor College of Medicine), Caspase 3 antibody (1:1000) (CellSignaling), VCP polyclonal antibody (1:200) (Santa Cruz Biotechnology)or anti-β-actin (1:10,000) or GAPDH antibodies (Sigma Aldrich) at 4° C.overnight; washed three times with 0.1% Tween 20-TBS; and incubated in ahorseradish peroxidase-linked secondary antibody (1:2,000) (CellSignaling) for 1 h at room temperature. The membrane was washed threetimes with 0.1% Tween 20-TBS, and the immunoreactive bands were detectedby using enhanced chemiluminescent (ECL) plus reagent kit (ThermoScientific). Western blots were quantified using ImageJ software (NIH,Bethesda) by comparing the expression of the protein in question to theexpression of the loading control.

In Silico Analyses

FSTL1 promoter analyses were performed using CONFAC (Karanam and Moreno,2004) software and confirmed using TRANSFAC analysis software (Matys etal., 2006). MiR-198 3′UTR target prediction was performed usingTargetScan software as previously described (Lewis et al., 2005), andconfirmed using PicTar and miRBase software. Conservation of miR-198binding sites in the PBX-1 and VCP 3′UTRs across species was determinedusing TargetScan (Friedman et al., 2009). RNA22 (Miranda et al., 2006)was used to predict full-length target sites for miR-198 within thecoding region of the MSLN gene. ImageJ software (NIH) (Rasband,1997-2011) was used to quantify western blot and anchorage independentgrowth assay images.

The promoter region of FSTL1 was analyzed as follows: FSTL1accessions/RefSeq ID was used by the web-based computer programConserved Transcription Factor Binding Site Finder (CONFAC) to get theMouse orthologs from ENSEMBL Mart databases and the Genomic sequencesfrom the UCSC Genome database. A pairwise BLAST on the Human-Mouseorthologous genes was then conducted to get the conserved parts whichwere then submitted to the MATCH program. The output from MATCH wasconsolidated into a set of reports which show the various ConservedTranscription Factor Binding Sites in the promoter regions of the genes,revealing several putative transcription factor binding sites in thesequence analyzed (FIG. 10).

Transient Transfection and Stable Cell Line Selection

Transfections were performed with Lipofectamine 2000 (Invitrogen)according to the manufacturer's instruction. For shRNA transfectionexperiments, a plasmid encoding MSLN shRNA (TR311377, Origene), OCT-2shRNA (TF310276) or PBX-1 shRNA (TG310592) and a control plasmidencoding GPF shRNA (TR30003) were used. Transfections with miRNAprecursors were performed using siPORT Transfection reagent and eithermiR-198 or scrambled control precursors (Ambion) according to themanufacturer's instruction.

MSLN-overexpressing stable cells (or empty vector controls) wereselected in MIA PaCa-2 cells or HPDE cells (MIA-MSLN, HPDE-MSLN) orvector (MIA-V, HPDE-V) using retrovirus vectors expressing puromycinresistance (Origene) with 0.5 μg/mL of puromycin added into the mediumas previously described (Zhang et al.). Stable cells overexpressingmiR-198 or vector control were generated in AsPC-1 (AsPC-1-miR-198,AsPC-1-miR-Ctrl), MIA-MSLN (MIA-MSLN-miR-198, MIA-MSLN-miR-Ctr1), MIA-V(MIA-V-miR-198, MIA-V-miR-Ctr1), and HPDE cells (HPDE-miR-198,HPDE-miR-Ctrl) using the Lenti-miR miRNA Precursor Clone Collection(SBI). The miRZip Lentivector collection from SBI was used to stablyknock down miR-198 in MIA-PaCa2 (MIA-V-Zip-198, MIA-V-Zip-Ctrl) and HPDE(HPDE-Zip-198, HPDE-Zip-Ctrl) cells.

miRNA and mRNA Extraction and Reverse-Transcription

Total miRNAs of tissues and cultured cells were extracted and purifiedusing mirVana miRNA Isolation kit (Applied Biosystems/Ambion) followingthe manufacturer's instructions. Five microliters of RNA were directlyconverted to cDNA with the QuantiMir™ RT System (SBI System Biosciences,Mountain View, Calif.). Total mRNA was extracted using the RNAqueous RNAIsolation kit (Applied Biosystems/Ambion). Two micrograms of RNA wereconverted to cDNA using the iQ SYBR Green supermix and iScript cDNAsynthesis kits purchased from Bio-Rad.

Real-Time RT-PCR

Differential expression of 95 miRNAs chosen for their potential roles incancer, cell development, and apoptosis was analyzed by RT-PCR using theQuantiMir System from SBI System Biosciences in both MIA-V and MIA-MSLNcells. The array plate also included the U6 transcript as anormalization signal. cDNAs from different cell lines and tissue sampleswere mixed with SYBR® Green Mastermix (Bio-Rad Laboratories) plus theuniversal reverse primer. Specific primers (1 μl) were added to eachwell of the qPCR plate. The miRNA sequences and primer sequences used inRT-PCR are listed in FIG. 9. Expression levels of each mature miRNA wereevaluated using comparative threshold cycle (Ct) method as normalized tothat of U6 (2-ΔCt). The expression level of miR-198 was measured in allsubsequent experiments using the same method. Real-time RT PCR wasperformed as previously described for MSLN, ZEB1, PBX-1, and VCP (Li etal., 2008). The primer sequences used are as follows:

for human MSLN (s = sense, a = antisense): [SEQ ID NO. 103]5′-CTCAACCCAGATGCGTTCTCG-3′ (s) and [SEQ ID NO. 104]5′-AGGTCCACATTGGCCTTCGT-3′ (a); for FSTL1: [SEQ ID NO. 105]5′-TCAGCATGACAGACCTCCAG-3 (s) and [SEQ ID NO. 106]5′-TCCCAGAAACTCCATCCAAG-3′ (a); for PBX-1: [SEQ ID NO. 107]5′-TGAGCGTGCAGTCACTCAATG-3′(s) and [SEQ ID NO. 108]5′-CGAGTCCATCACTGTATCCTCC-3′ (a); for ZEB1: [SEQ ID NO. 109]5′-TGCACTGAGTGTGGAAAAGC-3′ (s) and [SEQ ID NO. 110]5′-TGGTGATGCTGAAAGAGACG-3′ (a).

NF-κB Inhibitor Assays

MIA-MSLN and MIA-V control cells were treated with 0, 12.5 or 25 μM ofthe IKK inhibitor Wedelolactone (Calbiochem). The cells were collectedafter 72 h and protein, RNA, and miRNA extracts were prepared andassayed accordingly.

Anchorage Independent Growth Assay

A soft agar assay for colony formation was performed by coating 35 mmdishes with a combination of 0.5% base agar and 0.7% top agar. Base agarpreparation: 1% LMP Agarose (DNA grade) (Baylor College of Medicine) wasmelted in 1× incomplete DMEM, microwaved, and cooled to 40° C. in awater bath. Equal volumes of 20% FBS DMEM pre-warmed to 40° C. in waterbath were mixed to give 0.5% Agar+10% DMEM, of which 1.5 ml/35 m Petridish was added and allowed to set at room temperature.

Top agar preparation: 0.7% LMP Agarose (DNA grade) in 1×DMEM wasprepared in the same manner as above. Cells were trypsinized and dilutedto 0.1×106 cells/ml, in the 20% FBS DMEM; 5,000 cells/dish were platedin duplicate. The plates were then incubated at 37° C. in a humidifiedincubator for 15 days. Following incubation, the plates were stainedwith 0.5 ml of 1% MTT for 2 h, and colonies were photographed using adissecting microscope. Images were analyzed using IMAGEJ software.

Apoptosis Assay

Apoptosis induction was determined using western blotting for Caspase 3cleavage and confirmed by labeling DNA breaks using a terminaldeoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay withthe APO-DIRECT Kit (BD Biosciences) following 96 h of treatment (24 hserum starvation followed by 72 h of treatment with 20 ng/ml of TNF-α).Briefly, cells were plated in either 10% FBS media or serum-starved for96 h, at which time 1×10⁶ cells were harvested using trypsin-EDTA. Aftercentrifugation, the pellets were washed twice with PBS, then resuspendedin 1% paraformaldehyde in PBS and incubated on ice for 45 minutes. Afterseveral washes, the cell pellet again was resuspended in PBS/ethanol,incubated on ice for 30 minutes, and stored at −20° C. Cell samples werethen pelleted, washed, and resuspended in 50 μL of DNA labeling solutioncontaining a reaction buffer, terminal deoxynucleotidyl transferaseenzyme, FITC-labeled deoxyuridine triphosphate nucleotides, anddistilled water. After a 60-minute incubation at 37° C., cells wereagain washed, pelleted, and resuspended in 500 μl of propidiumiodine/RNase staining buffer. Samples were then analyzed by flowcytometry (fluorescence-activated cell sorting) using a FACSCalibur(Becton Dickinson, Franklin Lakes, N.J.). Data was further analyzedusing the software FLOWJOW ver. 6.1.1 (Tree Star Inc.).

Cell Proliferation Assay

Cells were seeded in 96-well plates (2×10³ cells per well) and serumstarved for 24 h prior to differential FBS additions.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasused to measure cell proliferation as previously described (Bharadwaj etal., 2011b).

Cell growth was assessed using MTT at 2, 3, 4, 5 and 6 days afterreleasing from starvation (not all data shown). Briefly, twentymicroliters of MTT reagent mixed with 100 μL of growth medium was addedto each well and incubated at 37° C. for 2 h. Cells were then lysedovernight using 100 μl of lysis buffer consisting of 20% SDS in 1:1dimethyl formamide; H₂O solution. Absorbance was recorded at 520 nm withan EL-800 universal microplate reader (Bio-Tek Instruments). Relativeproliferation was calculated by dividing the reading by a Day 0 readingtaken prior to serum starvation.

Migration, Invasion, and Wound Healing Assays

Cell migration was determined with a modified Boyden chamber assay asdescribed previously (Li et al., 2008). For invasion assays, Matrigelbasement membrane matrix (BD Biosciences) was used by adding 100 ul of 1mg/ml Matrigel to the migration chamber inserts prior to plating of thecells (Li et al., 2008, Bharadwaj).

An invasion assay was performed as described for the migration assay.For invasion, Matrigel basement membrane matrix (BD Biosciences) wasused by adding 100 μl of 1 mg/ml Matrigel to the migration chamberinserts prior to plating of the cells.

For wound healing assay, wounds were created in confluent monolayer ofcells with a sterile pipette tip in either 0%, 0.2%, 2%, or 5% FBSmedia, and wound healing was observed within the scrape line atdifferent time points; representative fields for each cell line werephotographed (Li et al., 2008, Bharadwaj).

Luciferase Reporter Constructs

For MSLN, six constructs were generated: 400 or 600 bp segmentscontaining the WT sequence or specific 3-nucleotide mutations in theseed region of each miR-198 binding site were synthesized by GenScriptand subcloned into the pEZX-MT01 3′UTR construct from Genecopoeia atXbal and BamH1 restriction sites such that they were inserted after theluciferase gene (FIG. 10). The same process was used for the VCPreporter constructs containing the full-length VCP 3′UTR (FIG. 10).

A luciferase construct containing the first 491 bases of the PBX-1 3′UTRwas purchased from Switchgear Genomics. Two mutant PBX-1 3′UTR reporterswere subsequently generated using the Stratagene QuickchangeSite-directed Mutagenesis kit, with the WT miR-198 binding site seedregion either altered from TCTGGA to AAGCTT (a HindIII restriction site)or deleted entirely (FIG. 10).

Dual-Luciferase Assays

Dual luciferase assays were performed according to the manufacturer'sinstruction. Luciferase expression analysis was performed at 24 h usingthe Dual-luciferase kit (Promega) and Sirius Luminometer (BertholdDetection System). Results are presented as the ratio of firefly toRenilla luciferase activities, normalized to controls.

PBX-1 Luciferase Reporters

The PBX-1 3′UTR region was cloned into the pSGG-3UTR vector (based onpGL4.11 vector with a MCS added after luciferase) after the reportergene, firefly luciferase, whose expression is regulated by the SV40promoter. A separate reporter for renilla luciferase under the controlof the CMV promoter was utilized to normalize for transfectionefficiency. The identity of the 3′UTR sequence including the predictedmiR-198 binding site was confirmed using the following primers: Fwd:CGTGAATTCTCACGGCTTCC [SEQ ID NO. 111], Rev: GCATCACAAATTTCACAAATAAAGC.[SEQ ID NO. 112]

The mutagenesis reactions were performed using the StratageneQuickchange Site-directed Mutagenesis kit from Agilent Technologies,according to the manufacturer's instructions. Briefly, Stratagenesoftware was used to generate the following primers introducing thedesired mutation or deletion, respectively:

5′-GGGATGCTATTTCAGCCAAAAGCTTCACTTCTTTATACTCTCTTCC-3′ (sense) [SEQ ID NO.113], 5′-CCTACGATAAAGTCGGTTTTCGAAGTGAAGAAATATGAGAGAAGGG-3′ [SEQ ID NO.114], (antisense), and 5′-CTTCTTTGGGATGCTATTTCAGCCAATACTTCTTTATACTCTC-3′(sense) [SEQ ID NO. 115], and5′-GAGAGTATAAAGAAGTATTGGCTGAAATAGCATCCCAAAGAAG-3′ (antisense) [SEQ IDNO. 116]. PCR amplification of the mutated plasmids was performedaccording to manufacturer specifications and followed by DpnI digestionof the parental methylated vectors. The plasmids were then transformedinto XL-10 Gold ultracompetent cells and plated on LB-Amp plates.Individual colonies were then grown overnight and plasmids were isolatedusing the QIAPrep Spin Miniprep Kit (QIAGEN) according to themanufacturer's instructions. The plasmids were sequenced to confirm theidentity of the nucleotide sequence using the following primers,specific for the 3′UTR cloned sequence in the vector:5′-CGTGAATTCTCACGGCTTCC-3′ (sense) [SEQ ID NO. 117], and5′-GCATCACAAATTTCACAAATAAAGC-3′ (antisense) [SEQ ID NO. 118].

MSLN CDS Mutant Constructs and Assay

A MSLN ORF clone was purchased from Origene (RC202532). Site-directedmutagenesis was used to generate constructs containing silent mutationsin each of the three predicted miR-198 binding sites within the CDS ofthe MSLN gene (FIG. 13). MiR-198 precursors or a scrambled miRNA controlwere co-transfected into COS-7 cells along with either the WT MSLN ORFconstruct or each of the single, double, or triple mutants. Cell lysateswere collected for western blot analysis. RNA and miRNA were collectedfor real-time RT-PCR analysis.

The primers used to generate mutations in each of the three predictedbinding sites for miR-198 within the MSLN CDS were as follows:

Site 1: 5′-CCTGGACGCCCTCCCACTAGATCTGCTGCTATTCTCA-3′ (s) [SEQ ID NO. 119]and 3′-GGACCTGCGGGAGGGTGATCTAGACGACGATAAGGAGT-5′ (a) [SEQ ID NO. 120],

Site 2: 5′-GCCTGCTGCCCGTGGTAGGACAGCCCATCATCCG-3′ (s) [SEQ ID NO. 121]and 3′-CGGACGACGGGCACCATCCTGTCGGGTAGTAGGC-5′ (a) [SEQ ID NO. 122];

Site 3: 5′-CGAGTCTGTGATCCAGCACTTAGGATACCTCTTCCTCAAGATGA-3′ (s) [SEQ IDNO. 123] and 3′-GCTCAGACACTAGGTCGTGAATCCTATGGAGAAGGAGTTCTACT-5′ (a) [SEQID NO. 124];

and corresponded to the regions of the MSLN CDS depicted in supplementalFIG. 13. The PCR conditions and transformations were performed asdescribed above. Double and triple mutants were generated serially, andall mutations were verified by sequencing.

Example 10

Additional in silico analysis also revealed 17 putative binding sitesfor the negative transcription factor ZEB1 in the miR-198 promoter. ZEB1is a crucial epithelial-to-mesenchymal transition (EMT) activator inhuman colorectal and breast cancer (Burk et al), and has been linked toincreased EMT and chemoresistance in pancreatic cancers (Wang et al).ZEB1 also directly suppresses transcription of and is involved in areciprocal regulatory loop with miRNAs in the miR-200 family (Burk etal).

Also examined here was whether ZEB1 was also acting to repress miR-198expression. While MIA-PaCa2 cells have been previously reported to havea relatively high level of ZEB1 expression (Wellner et al), it is alsoknown that constitutive activation of NF-κB in breast cancer cells andPC cells leads to induction of ZEB1 expression (Maier et al, Chua etal). In accordance with these previous findings, here it was found thatZEB1 is further induced in MIA-PaCa2 cells as a result of MSLNoverexpression (Figure III.3-A). Transfecting MSLN-specific shRNAs inMIA-MSLN cells led to reduced expression of ZEB1 both at the mRNA level(70% reduction) (p<0.05) (Figure B) and at the protein level (˜50%reduction) (Figure C). The involvement of NF-κB activation in ZEB1induction was confirmed using Wedelolactone, which completely blockedZEB1 expression in MIA-MSLN cells (Figure D). In an embodiment of thedisclosure, ZEB1 might be contributing to miR-198 repression, andoverexpression of ZEB1 in PC cells may contribute to the decrease inmiR-198 expression from that of normal tissues. At a certain thresholdlevel, such as that induced through MSLN overexpression, ZEB1 levelsbecome sufficient to completely repress miR-198 expression. Consistentwith this, immortalized HPDE control cells, which have the highestmiR-198 levels (Figure III-1-C), have no detectable ZEB1 expression(Figure E). Furthermore, miRNAs in the miR-200 family previouslyreported to be downregulated by ZEB1, including miR-141 and miR-200c(Burk et al) were in fact further downregulated following MSLNoverexpression in MIA-MSLN cells from the already repressed levels ofMIA-V cells (Figure A), indicating that the upregulation of ZEB1 didhave further strong functional effects even in these highly expressingZEB1 cells. To confirm the regulatory effects of ZEB1 expression onmiR-198 levels, ZEB1-specific shRNAs were transfected into MIA-MSLNcells, which resulted in a partial, ˜6.5 fold increase in miR-198expression compared to controls. Cells transfected with a less effectiveshRNA that showed only a partial reduction in ZEB1 protein expressionalso showed a partial (2-fold) increase in miR-198 expression (FigureF). Conversely, further overexpressing ZEB1 in MIA-PaCa2 cells reducedmiR-198 expression ˜9 fold (p<0.05) (Figure III.3-G). While the aboveresults implicated ZEB1 in the regulation of miR-198, ZEB1 modulationappeared to only partially recapitulate the effects of NF-κB or MSLNmodulation on miR-198, with only a ˜6.5 fold increase in miR-198expression after ZEB1 modulation compared to a ˜12-18-fold changethrough MSLN or NF-κB modulation. Furthermore, some PC cell lines withhigh MSLN and low miR-198 levels, such as HPAF-II and BxPC3, have beenpreviously reported to have low ZEB1 expression levels (Wellner et all),indicating that MSLN-mediated ZEB1 induction may only be occurring incertain cell lines, and that, while ZEB1 can in certain cases repressmiR-198, other mechanisms of MSLN-mediated miR-198 repression may playimportant roles. ZEB1 may therefore contribute along with OCT-2 as arepressor of miR-198.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

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What is claimed is:
 1. A method of treating ovarian cancer in anindividual, comprising the step of delivering to the individual aneffective amount of a composition that increases the level ofmicroRNA-198 molecules in cancer cells of the individual.
 2. The methodof claim 1, wherein the composition comprises microRNA-198, amicroRNA-198 mimic, or a modified microRNA-198.
 3. The method of claim1, wherein the composition is administered by a viral vector, anon-viral vector or a combination thereof.
 4. The method of claim 1,wherein the composition is administered locally, systemically, or acombination thereof.
 5. The method of claim 1, wherein the compositionis administered by a liposome, a viral vector, nanocarrier, or amicrocarrier.
 6. The method of claim 1, wherein the composition isdelivered in multiple cycles of treatment.
 7. The method of claim 1,wherein increasing the levels of microRNA-198 molecules causesimprovement by inhibiting migration, invasion, proliferation, tumorgrowth, metastatic potential, tumorigenesis or a combination thereof ofthe cancer.
 8. The method of claim 1, wherein the individual is furtherprovided one or more additional anti-cancer therapies.
 9. The method ofclaim 8, wherein the additional anti-cancer therapy compriseschemotherapy, radiotherapy, immunotherapy, gene therapy, surgery,non-microRNA-198 microRNA, siRNA or a combination thereof.
 10. Themethod of claim 1, further comprising the step of determining the levelsof MSLN, ZEB 1, OCT-2, PBX-1, VCP, or combinations thereof in the cancercells of the individual.
 11. The method of claim 1, wherein themiRNA-198 comprises SEQ ID NO:1.
 12. The method of claim 1, wherein thecancer cells of the individual express MSLN, ZEB1, OCT-2, PBX-1, VCP, orany combination thereof.
 13. A method of inhibiting proliferation andmetastatic potential of at least one ovarian cancer cell in anindividual, comprising delivering to the individual an effective amountof a composition that increases the levels of microRNA-198 molecules inthe cancer cell.
 14. The method of claim 13, wherein the composition isadministered by a viral vector, a non-viral vector or a combinationthereof.
 15. The method of claim 13, wherein the composition isadministered locally, systemically, or a combination thereof.
 16. Themethod of claim 13, wherein the agent comprises microRNA-198.
 17. Themethod of claim 13, wherein the composition comprises modifiedmicroRNA-198 oligonucleotide.
 18. The method of claim 13, wherein thecomposition comprises a microRNA-198 mimic.
 19. The method of claim 13,wherein the composition is administered by a liposome, a viral vector,or a microcarrier.
 20. The method of claim 13, wherein the individual isfurther provided one or more additional anti-cancer therapies.
 21. Themethod of claim 20, wherein the additional anti-cancer therapy compriseschemotherapy, radiotherapy, immunotherapy, gene therapy, surgery,non-microRNA-198 microRNA, siRNA, or a combination thereof.
 22. Themethod of claim 20, wherein the additional anti-cancer therapy comprisesgemcitabine, 5-fluorouracil, Irinotecan, Oxaliplatin, cyclophosphamide,etoposide, Ifosfamide, irinotecan, doxorubicin, Melphalan, Vinorelbine,paclitaxel, Capecitabine, Cisplatin, or a combination thereof.
 23. Themethod of claim 13, wherein the agent is delivered in multiple cycles oftreatment.
 24. The method of claim 13, wherein the cancer cell expressesMSLN, ZEB1, OCT-2, PBX-1, VCP, or any combination thereof.
 25. Themethod of claim 13, further comprising the step of determining thelevels of MSLN, ZEB1, OCT-2, PBX-1, VCP, or combinations thereof in thecancer cells of the individual.